Polyamide-oligonucleotide derivatives, their preparation and use

ABSTRACT

Polyamide-oligonucleotide derivatives of the formula
 
F[(DNA-Li) q (PNA-Li) r (DNA-Li) s (PNA) t ] x F′
 
wherein q, r, s, t are, independently of one another, zero or 1, where the total of two or more adjacent q, r, s and t≧2; x is 1 to 20; DNA is a nucleic acid such as DNA or RNA or a known derivative thereof; Li is a covalent linkage between DNA and PNA, where the covalent linkage comprises a bond or an organic radical with at least one atom from the series consisting of C, N, O or S; PNA is a polyamide structure which contains at least one nucleotide base which is different from thymine; and F and F′ are end groups and/or are linked together by a covalent bond, and the physiologically tolerated salts thereof, a process for their preparation and their use as pharmaceutical, as gene probe and as primer, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a divisional of U.S. patent application Ser. No.09/793,146, filed Feb. 27, 2001 now U.S. Pat. No. 6,919,441, which is acontinuation of U.S. patent application Ser. No. 08/982,008, filed Dec.1, 1997 now abandoned, which is a Continuation-in-Part of U.Sapplication Ser. No. 08/402,838 filed Mar. 13, 1995, now abandoned,priority to both of which is claimed, and the disclosures of which arehereby incorporated.

The present invention relates to novel polyamide-oligonucleotidederivatives with valuable physical, biological and pharmacologicalproperties. Their application relates to use as inhibitors of geneexpression (antisense oligonucleotides, ribozymes, senseoligonucleotides and triplex forming oligonucleotides), as probes fordetecting nucleic acids and as aids in molecular biology.

Oligonucleotides are finding increasing application as inhibitors ofgene expression (G. Zon, Pharmaceutical Research 5, 539 (1988); J. S.Cohen, Topics in Molecular and Structural Biology 12 (1989) MacmillanPress; C. Helene and J. J. Toulme, Biochimica et Biophysica Acta 1049,99 (1990); E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990)).Antisense oligonucleotides are nucleic acid fragments whose basesequence is complementary to that of an mRNA to be inhibited. Thistarget mRNA can be of cellular, viral or other pathogenic origin.Suitable cellular target sequences are, for example, those of receptors,cell-adhesion proteins, enzymes, immunomodulators, cytokines, growthfactors, ion channels or oncogenes. Inhibition of virus replication withthe aid of antisense oligonucleotides has been described, for example,for HBV (hepatitis B virus), HSV-1 and -2 (herpes simplex virus type Iand II), HIV (human immunodeficiency virus) and influenza viruses. Thisentails use of oligonucleotides which are complementary to the viralnucleic acid. Sense oligonucleotides are, by contrast, designed to havea sequence such that, for example, they bind (“trap”) nucleicacid-binding proteins or nucleic acid-processing enzymes and thusinhibit their biological activity (C. Helene and J. J. Toulme,Biochimica et Biophysica Acta 1049, 99 (1990)). Viral targets which maybe mentioned here are, for example, reverse transcriptase, DNApolymerase and transactivator proteins. Triplex-forming oligonucleotidesgenerally have the DNA as target and, after binding thereto, form atriple helix structure. Whereas in general the processing (splicingetc.) of the mRNA or translation thereof into protein is inhibited byantisense oligonucleotides, the transcription or replication of the DNAis inhibited by triplex-forming oligonucleotides (C. Helene and J. J.Toulme, Biochim. Biophys. Acta 1049 (1990) 99-125; E. Uhlmann and A.Peyman, Chemical Reviews 90, 543 (1990)). However, it is also possibleto bind single-stranded nucleic acids in a first hybridization with anantisense oligonucleotide to form a double strand, which then in asecond hybridization with a triplex-forming oligonucleotide forms atriplex structure. The antisense and triplex binding regions may in thiscase be accommodated either in two separate oligonucleotides or else inone oligonucleotide. Another application of synthetic oligonucleotidescomprises so-called ribozymes which destroy the target RNA as aconsequence of their ribonuclease activity (J. J. Rossi and N. Sarver,TIBTECH (1990) 8, 179; Castanetto et al., Critical Rev. Eukar. GeneExpr. (1992) 2, 331).

The compounds according to the invention can also be used in therapy inthe sense of aptamers. Aptamers are oligomeric nucleic acids or analogsthereof which bind with high affinity to proteins. The aptamers arefound by in vitro selection from a random mixture (Famulok and Szostak(1992) Angew. Chem. 104, 1001-1011) and this has been carried outsuccessfully for a thrombin-binding aptamer (Bock et al. (1992) Nature355, 564-566). The procedure for this can be such that the base sequenceof the aptamer is determined by screening an oligonucleotide mixture,and this base sequence is then transferred to polyamide-oligonucleotideanalogs. Another possibility comprises encoding the binding region ofthe aptamer, to facilitate identification, by a separate non-bindingpart of the molecule (Brenner and Lerner (1992) PNAS 89, 5381-5383).

In DNA diagnosis, nucleic acid fragments with suitable labeling are usedas so-called DNA probes for specific hybridization onto a nucleic acidto be detected. The specific formation of the new double strand is inthis case followed with the aid of the labeling, which is preferablynon-radioactive. It is possible in this way to detect genetic, malignantor viral diseases or diseases caused by other pathogens.

Oligonucleotides in their naturally occurring form have little or nosuitability for most of the said applications. They have to bechemically modified so that they satisfy the specific requirements. Foroligonucleotides to be employable in biological systems, for example forinhibition of virus replication, they must meet the followingrequirements:

-   1. They must have sufficiently high stability under in vivo    conditions, that is to say both in serum and intracellularly.-   2. Their properties must be such that they can pass through the cell    membrane and nuclear membrane.-   3. Under physiological conditions they must bind in a base-specific    manner to their target nucleic acid in order to display the    inhibitory effect.

Points 1 to 3 are not a requirement for DNA probes; however, theseoligonucleotides must be derivatized so that detection is possible, forexample by fluorescence, chemiluminescence, colorimetry or specificstaining (Beck and Köster, Anal. Chem. 62, 2258 (1990)). The chemicalmodification of the oligonucleotides usually takes place by appropriatemodification of the phosphate backbone, ribose unit or the nucleotidebases (J. S. Cohen, Topics in Molecular and Structural Biology 12 (1989)Macmillan Press; E. Uhlmann and A. Peyman, Chemical Reviews 90, 543(1990)). Another frequently used method is to prepare oligonucleotide 5′conjugates by reaction of the 5′-hydroxyl group with appropriatephosphorylation reagents. If, on the other hand, all the internucleotidephosphate residues are modified there is often a drastic change in theproperties of the oligonucleotides. For example, the solubility ofmethyl phosphonates in aqueous medium is greatly reduced, whileall-phosphorothioate oligonucleotides often act in anon-sequence-specific manner.

There have recently been descriptions of polyamide-nucleic acidderivatives (Michael Egholm, Peter E. Nielsen, Rolf H. Berg and OleBuchardt, Science 1991, 254, 1497-1500; WO 92/20702; M. Egholm et al.Nature (1993) 365, 566-568; P. Nielsen, (1994) Bioconjugate Chem. 5,3-7) which bind to complementary target sequences (DNA or RNA) withhigher affinity than natural oligonucleotides. These so-called peptideor polyamide nucleic acids (PNA) are DNA-analogous compounds in whichthe deoxyribose phosphate skeleton has been replaced by a polyamideoligomer. These compounds have the advantage compared with naturaloligonucleotides that they are very stable in serum. However, on theother hand, they have the following disadvantageous properties:

-   (1) The amount taken up in cells is zero or undetectable. However,    since antisense or triplex-forming oligonucleotides are able to    display their activity only in the cell, the PNAs as such are    unsuitable for inhibition of gene expression in vivo.-   (2) The PNAs tend to aggregate in aqueous solution, that is to say    also under physiological conditions. Their solubility in aqueous    buffer is therefore low and they are unavailable for hybridization    to complementary sequences.-   (3) The PNAs additionally have high affinity for various materials    such as ®Sephadex (from Pharmacia) or ®Bond Elut (from Varian) used    to purify the oligomers, so that the PNAs can often be isolated only    in poor yields.-   (4) Another serious disadvantage of the PNAs is that they do not    bind in an unambiguous orientation to complementary nucleic acids.    The sequence specificity is therefore reduced by comparison with    natural oligonucleotides. Whereas natural nucleic acids generally    hybridize to complementary nucleic acids in the antiparallel    orientation, PNAs may bind both in the antiparallel and in the    parallel orientation.-   (5) WO 92/20702 mentions an oligonucleotide-PNA conjugate    (T)₇(5′-L-N)(t)₆-Ala (FIG. 25; substitute sheet), where (T)₇ is a    natural heptathymidylate oligonucleotide which is linked via its    5′-O-phosphate and 4-hydroxybutyric acid (L) to the primary amino    group (N) of a PNA-hexathymidylate (t)₇ and alanine (Ala). Neither    the synthesis of this compound nor any properties have been    described.-   (6) PNAs show highly cytotoxic properties in the μmolar range in    cell culture experiments.

The orientation of the base-pairing nucleic acid strands is defined asfollows: (cf. Egholm et al.; Nature 365 (1993) 566-568).

A) 5′ ---------- 3′ DNA ap Duplex ap = antiparallel 3′ ---------- 5′ DNAB) 5′ ---------- 3′ DNA p Duplex p = parallel 5′ ---------- 3′ DNA C) 5′---------- 3′ DNA ap Duplex (DNA · PNA) C ----------- N PNA D) 5′---------- 3′ DNA p Duplex (DNA · PNA) N ----------- C PNA E) C----------- N PNA 5′ ---------- 3′ DNA (Pu) ap · ap triplex (DNA · DNA ·PNA) 3′ ---------- 5′ DNA      Pu = purine-rich strand F) N -----------C PNA 5′ ---------- 3′ DNA (Pu) ap · p triplex (DNA · DNA · PNA) 3′---------- 5′ DNA G) N ----------- C PNA 5′ ---------- 3′ DNA (Pu) ap ·p triplex (PNA · DNA · PNA) C ----------- N PNA H) C ----------- N PNA5′ ---------- 3′ DNA (Pu) ap · ap triplex (PNA · DNA · PNA) C----------- N′ PNA I) N ----------- C PNA 5′ ---------- 3′ DNA (Pu) p ·p triplex (DNA · DNA · PNA) N ----------- C′ PNA K) C ----------- N PNA5′ ---------- 3′ DNA (Pu) p · ap triplex (DNA · DNA · PNA) N -----------C PNA where 5′ means the 5′ end of an oligonucleotide, 3′ means the 3′end of an oligonucleotide, N means the amino terminus of a PNA C meansthe carboxyl terminus of a PNA.Cases A)-D) are examples of the types of orientation which are possiblein principle for the antisense oligomers. Cases E)-F) show possibilitiesfor triplex formation on single-stranded or double-stranded nucleicacids.

It is moreover possible for two of the PNA or DNA single strands to belinked together. For example, in E) the N terminus of the PNA can belinked to the 5′ end of the DNA, or in F) the C terminus of the PNA canbe linked to the 5′ end of the DNA.

The object of the invention therefore was to preparepolyamide-oligonucleotide derivatives in which the abovementioneddisadvantages are eliminated.

The invention relates to polyamide-oligonucleotide derivatives of theformula IF[(DNA-Li)_(q)(PNA-Li)_(r)(DNA-Li)_(s)(PNA)_(t)]_(x)F′  (I)wherein

-   q, r, s, t are, independently of one another, zero or 1, where the    total of two or more adjacent q, r, s and t≧2;-   x is 1 to 20, preferably 1 to 5, particularly preferably 1;-   DNA is a nucleic acid such as DNA or RNA or a known derivative    thereof;-   Li is a covalent linkage between DNA and PNA, where the covalent    linkage comprises a bond or an organic radical with at least one    atom from the series consisting of C, N, O or S;-   PNA is a polyamide structure which contains at least one nucleotide    base which is different from thymine; and-   F and F′ are end groups and/or are linked together by a covalent    bond (cyclic compounds),    and the physiologically tolerated salts thereof.

Particular mention may furthermore be made of polyamide-oligonucleotidederivatives of the formula I in which x is 1 and, at the same time,

-   q=r=1 and s=t=zero or-   r=s=1 and q=t=zero or-   q=r=s=1 and t=zero or-   r=s=t=1 and q=zero.

Preferred compounds have the formulae Ia and Ib

in which

-   x is 1 to 20, where when x>1 r=s=1 and, at the same time, q=t=zero    and o=n=zero to 5;-   q, r, s, t are, independently of one another, zero or 1, where the    total of two or more adjacent q, r, s and t≧2;-   R² is hydrogen, hydroxyl, C₁-C₁₈-alkoxy, preferably C₁-C₆-alkoxy,    halogen such as F or Cl, preferably F, azido or amino;-   B is, independently of one another, a base customary in nucleotide    chemistry, for example natural bases such as adenine, cytosine,    thymine, guanine, uracil, inosine or unnatural bases such as, for    example, purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine,    N⁴,N⁴-ethanocytosine, N⁶,N⁶-ethano-2,6-diaminopurine,    pseudoisocytosine, 5-methylcytosine, 5-fluorouracil,    5-(C₃-C₆)-alkynyluracil, 5-(C₃-C₆)-alkynylcytosine or the prodrug    forms thereof, and the “curved bracket” indicates that R² and the    adjacent substituent can be in the 2′ position and 3′ position or    else conversely in the 3′ position and 2′ position;-   Nu is a radical of the formulae IIa or IIb

-   -   in which    -   R² and B are as defined above;    -   U is hydroxyl, mercapto, C₁-C₁₈-alkyl, preferably C₁-C₈-alkyl,        C₁-C₁₈-alkoxy, preferably C₁-C₈-alkoxy, C₆-C₂₀-aryl, preferably        C₆-C₁₂-aryl, C₆-C₁₄-aryl-C₁-C₈-alkyl, preferably        C₆-aryl-C₁-C₄-alkyl, NHR³ or NR³R⁴, and

-   R³ is C₁-C₁₈-alkyl or C₁-C₄-alkoxy-C₁-C₄-alkyl, preferably    C₁-C₈-alkyl or C₁-C₄-alkoxy-C₁-C₄-alkyl, particularly preferably    C₁-C₄-alkyl or methoxyethyl and

-   R⁴ is C₁-C₁₈-alkyl, preferably C₁-C₈-alkyl and particularly    preferably C₁-C₄-alkyl, or

-   R³ and R⁴ is, together with the nitrogen atom carrying them, a    5-6-membered heterocyclic ring which can additionally contain    another hetero atom from the series consisting of O, S, N, such as,    for example, morpholine;

-   V is oxy, thio or imino;

-   W is oxo or thioxo;

-   Y is oxy, thio, methylene or imino;

-   m is zero to 20;

-   o is zero to 20;

-   D is a radical of the formula III

-   -   in which B is as defined above;

-   D′ is a radical of the formula IV

-   -   in which B is as defined above;

-   n is zero to 20;

-   p is zero to 20;

-   Li₁, Li₂, Li₃ and Li₄ are each, independently of one another, a    structure of the formula V    [(V′)-(G)-(G′)]_(ε)  (V)    -   where, independently of one another,    -   ε is 1 to 5, preferably 1-2,    -   V′ is oxygen, NH, a bond or a radical of the formula VI

-   -   in which    -   U, V, W and Y are as defined above;

-   G can be C₁-C₁₂-alkanediyl, preferably C₁-C₆-alkanediyl, where    alkanediyl can optionally be substituted by halogen, preferably F or    chlorine, amino, hydroxyl, C₁-C₁₈-alkyl, preferably C₁-C₆-alkyl,    C₁-C₁₈-alkoxy, preferably C₁-C₆-alkoxy, C₆-C₁₄-aryl, preferably    C₆-aryl, or C₆-C₁₄-aryl-C₁-C₁₈-alkyl, preferably    C₆-aryl-C₁-C₄-alkyl; C₆-C₁₄-aryl-di-C₁-C₁₂-alkanediyl, preferably    C₆-aryl-di-C₁-C₄-alkanediyl, or a group of the formula    (CH₂CH₂O)_(δ)CH₂CH₂ in which δ can be 1 to 11, preferably 1 to 7; or    a bond; and

-   G′ is oxy, thio, imino, —C(O)—, —C(O)NH—, a bond or a radical of the    formula VI in which U, V, W and Y are as defined above; and

-   F and F′ are linked by a bond (cyclic compounds) and/or

-   F is R⁰— (A)_(k)-V— and

-   F′ in formula Ia is -(Q)₁-R¹ and in formula Ib is V¹-(A)₁-R¹,    -   where    -   R⁰ is hydrogen, C₁-C₁₈-alkanoyl, preferably C₈-C₁₈-alkanoyl,        C₁-C₁₈-alkoxycarbonyl, C₃-C₈-cycloalkanoyl, C₇-C₁₅-aroyl,        C₃-C₁₃-heteroaroyl or a group which favors intracellular uptake        of the oligomer or serves as labeling of a DNA probe or, in the        hybridization of the oligomer onto the target nucleic acid,        attacks the latter with binding, crosslinking or cleavage; or    -   if k is zero, R⁰ is hydrogen or together with V is a radical of        the formula VII

-   -    in which    -   Z and Z′ are, independently of one another, hydroxyl, mercapto,        C₁-C₂₂-alkoxy, preferably C₁₂-C₁₈-alkoxy, C₁-C₁₈-alkyl,        preferably C₁₂-C₁₈-alkyl, C₆-C₂₀-aryl, preferably C₆-C₁₆-aryl,        C₆-C₁₄-aryl-C₁-C₁₈-alkyl, preferably C₆-aryl-C₁-C₄-alkyl,        C₁-C₂₂-alkylthio, preferably C₁₂-C₁₈-alkylthio, NHR³, NR³R⁴, or        a group which favors intracellular uptake of the oligomer or        serves as labeling of a DNA probe or, in the hybridization of        the oligomer onto the target nucleic acid, attacks the latter        with binding, crosslinking or cleavage, and in which        -   R³, R⁴, V and W are as defined above;    -   R¹ is hydrogen or Q^(o)        -   where R¹ is always only hydrogen when at the same time 1 is            zero and in formula Ia t is zero and s is 1 and Li₁ is a            structure of the formula V with V′=bond, G=bond, ε=1 and            G′=oxy, thio, imino or a radical of the formula VI with U=Z            or        -   in formula Ib q is 1 or q=r=zero and in F′=V¹-(A)₁-R¹ with            V¹=V,    -   A and Q are, independently of one another, the residue of a        natural or unnatural amino acid, preferably from the series        consisting of glycine, leucine, histidine, phenylalanine,        cysteine, lysine, arginine, aspartic acid, glutamic acid,        proline, tetrahydroisoquinoline-3-carboxylic acid,        octahydroindole-2-carboxylic acid, N-(2-aminoethyl)glycine;    -   Q^(o) is hydroxyl, OR′, NH₂, NHR″ with        -   R′=C₁-C₁₈-alkyl, preferably C₁₂-C₁₈-alkyl and            R″=C₁-C₁₈-alkyl, preferably C₁₂-C₁₈-alkyl,            C₁-C₁₈-aminoalkyl, preferably C₁₂-C₁₈-aminoalkyl,            C₁-C₁₈-hydroxyalkyl, preferably C₁₂-C₁₈-hydroxyalkyl;    -   V is as defined above;    -   V¹ is a bond or V, where in F′ only in formula Ib with q=zero        and r=1 V¹ is always a bond;    -   k is zero to 10;    -   l is zero to 10;        with the proviso that

-   a) if in the compound of the formula Ia t is zero and s is 1, and    Li₁ is (V′)-(G)-(G′) with V′=a compound of the formula VI,    G=C₂-C₁₂-alkylene and G′=CO, in F′=−(Q)₁-R¹ l is zero to 10 and R¹    is Q^(o);

-   b) if in the compound of the formula Ia s=t=zero, Li₂ is a bond;

-   c) if in the compound of the formula Ib t is zero and s is 1, Li₃ is    a bond;

-   d) if in the compound of the formula Ib s=t=zero, Li₄ is a bond;    where each nucleotide can be in its D or L configuration, and the    base can be in the α or β position.

Particularly preferred compounds of the formula Ia and Ib are those inwhich the base is located on the sugar in the β position,

-   x is 1 and-   q=r=1, s=t=zero or-   r=s=1, q=t=zero or-   q=r=s=1, t=zero or-   r=s=t=1, q=zero.

Especially preferred oligomers have the formulae Ia and Ib in which V′,V, Y and W have the meaning of thio, oxy, oxo or hydroxyl; these arevery particularly preferred if, in addition, R² is hydrogen.

Also especially preferred are oligomers of the formulae Ia and Ib withε=1, in which

-   Li₁, Li₄ are    -   a) a compound of the formula V in which V′=oxygen or compound of        the formula VI, G=C₁-C₁₀-alkylene, G′=—CONH—)    -   b) a compound of the formula V in which G, V′ is a bond and G′        is a compound of the formula VI with, preferably, U=V=W=Y=oxygen        or U=W=Y=oxygen and V=imino-   Li₂, Li₃ are    -   a) a compound of the formula V with V′=imino, G=C₁-C₁₀-alkylene        and G′=compound of the formula VI    -   b) a compound of the formula V with V′=imino, G and G′=bond    -   c) a compound of the formula V with V′=imino, G=C₁-C₁₀-alkylene        and G′=V with, preferably, U=V=W=Y=oxygen.

Very particularly preferred oligomers have the formulae Ia and Ib inwhich V′, V, Y and W have the meaning of thio, oxy, oxo or hydroxyl, R²is hydrogen, Li₁ has the meaning of —V′—[CH₂]_(n)C(O)NH— withV′=compound of the formula VI with U=V=W=Y=oxygen or Li₂ has the meaningof —HN—[CH₂]_(n)(G′)-, where n is 2 to 5 and G′ has the formula VI withU, V, W and Y=oxygen.

Additionally preferred oligomers of the formulae Ia and Ib are those inwhich V′, V, Y and W have the meaning of thio, oxy, oxo or hydroxyl, R²is hydrogen, Li₁ has the meaning of —O—[CH₂]_(n)C(O)NH— or Li₂ has themeaning of —HN—[CH₂]_(n)(G′)-, where n is 2 to 5 and G′ has the formulaVI with U, V, W and Y=oxygen, and q=zero and r=s=t=1.

Additionally preferred are oligomers of the formulae Ia and Ib in whichthe curved bracket means that R² is in the 3′ position (see formulaIIb). The preferred base in this case is adenine.

The invention is not confined to α- and β-D- and L-ribofuranosides, α-and β-D- and L-deoxyribofuranosides and corresponding carbocyclicfive-membered ring analogs but also applies to oligonucleotide analogswhich are composed of different sugar building blocks, for examplering-expanded and ring-contracted sugars, acyclic, ring-bridged or othersuitable types of sugar derivatives. The invention is furthermore notconfined to the derivatives, indicated by way of example in formula I,of the phosphate residue but also relates to known dephosphoderivatives.

The oligonucleotide part (DNA in formula I) can therefore be modifiedfrom the natural structure in a wide variety of ways. Examples of suchmodifications, which are introduced by methods known per se, are:

-   a) Modifications of the phosphate bridge

Examples which may be mentioned are: phosphorothioates,phosphorodithioates, methylphosphonates, phosphoramidates,boranophosphates, phosphate methyl esters, phosphate ethyl esters,phenylphosphonates. Preferred modifications of the phosphate bridge arephosphorothioates, phosphorodithioates and methylphosphonates.

-   b) Replacement of the phosphate bridge

Examples which may be mentioned are: replacement by formacetal,3′-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo,dimethylene sulfone, silyl groups. Replacement by formacetals and3′-thioformacetals is preferred.

-   c) Modifications of the sugar

Examples which may be mentioned are: α-anomeric sugars,2′-O-methylribose, 2′-O-butylribose, 2′-O-allylribose,2′-fluoro-2″-deoxyribose, 2′-amino-2′-deoxyribose, α-arabinofuranose,carbocyclic sugar analogs. The preferred modification is that by2′-O-methylribose and 2′-O-n-butylribose.

-   d) Modifications of the bases with do not alter the specificity of    the Watson-Crick base pairing

Examples which may be mentioned are: 5-propynyl-2′-deoxyuridine,s-propynyl-2′-deoxycytidine, 5-hexynyl-2′-deoxyuridine,5-hexynyl-2′-deoxycytidine, 5-fluoro-2′-deoxycytidine,5-fluoro-2′-deoxyuridine, 5-hydroxymethyl-2′-deoxyuridine,5-methyl-2′-deoxycytidine, 5-bromo-2′-deoxycytidine. Preferredmodifications are 5-propynyl-2′-deoxyuridine, 5-hexynyl-2′-deoxyuridine,5-hexynyl-2′-deoxycytidine and 5-propynyl-2′-deoxycytidine.

-   e) 3′-3′ and 5′-5′ inversions [for example M. Koga et al., J. Org.    Chem. 56 (1991) 3757]-   f) 5′- and 3′-phosphates, and 5′- and 3′-thiophosphates.

Examples of groups which favor intracellular uptake are variouslipophilic radicals such as —O—(CH₂)_(x)—CH₃ in which x is an integerfrom 6 to 18, —O—(CH₂)_(n)—CH═CH—(CH₂)_(m)—CH₃ in which n and m are,independently of one another, an integer from 6 to 12,—O—(CH₂CH₂O)₄—(CH₂)₉—CH₃, —O—(CH₂CH₂O)₈—(CH₂)₁₃—CH₃ and—O—(CH₂CH₂O)₇—(CH₂)₁₅—CH₃, but also steroid residues such as cholesterylor vitamin residues such as vitamin E, vitamin A or vitamin D and otherconjugates which utilize natural carrier systems such as bile acid,folic acid, 2-(N-alkyl-N-alkoxyamino)-anthraquinone and conjugates ofmannose and peptides of the appropriate receptors which lead toreceptor-mediated endocytosis of the oligonucleotides, such as EGF(Epidermal Growth Factor), bradykinin and PDGF (Platelet Derived GrowthFactor). By labeling groups are meant fluorescent groups, for example ofdansyl (=1-dimethylaminonaphthalene-5-sulfonyl), fluorescein or coumarinderivatives or chemiluminescent groups, for example of acridinederivatives, and the digoxigenin system detectable by ELISA, the biotingroup detectable by the biotin/avidin system or else linker arms withfunctional groups which permit subsequent derivatization with detectablereporter groups, for example an aminoalkyl linker which is reacted withan acridinium active ester to give the chemiluminescence probe. Typicallabeling groups are:

Oligonucleotide analogs which bind to or intercalate and/or cleave orcrosslink nucleic acids contain, for example, acridine, psoralen,phenanthridine, naphthoquinone, daunomycin or chloroethylaminoarylconjugates. Typical intercalating and crosslinking radicals are:

Examples which may be mentioned of NR³R⁴ groups in which R³ and R⁴ form,together with the nitrogen atom carrying them, a 5- to 6-memberedheterocyclic ring which additionally contains another hetero atom arethe morpholinyl and the imidazolidinyl radical.

The polyamide part (PNA in formula I) is composed of amide structureswhich contain at least one nucleotide base which is different fromthymine. Polyamide structures of this type are composed, for example, ofthe following building blocks a) to h), preferably a), in which f is 1to 4, preferably 1 or 2 and g is zero to 3, preferably zero to 2:

-   Hyrup et al.; J. Chem. Soc. Chem. Comm. 1993, 519

-   De Konig et al. (1971) Rec. Trav. Chim. 91, 1069

-   Huang et al. (1991) J. Org. Chem. 56, 6007

-   Almarsson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7518

-   Froehler et al. (1991) WO 93/10820

-   Froehler et al. (1991) WO 93/10820

-   Lewis (1993) Tetrahedron Lett. 34, 5697.

End groups for PNAs are described in the applications, filedsimultaneously, with the titles “PNA synthesis using an amino protectivegroup which is labile to weak acids” (HOE 94/F 060, DE-P 44 08 531.1)and “PNA synthesis using a base-labile amino protective group” (HOE 94/F059, DE-P 44 08 533.8).

Preferred polyamide structures are composed of structures according toa). The latter are particularly preferred when f is 1.

The preparation of polyamide-oligonucleotide derivatives of the formulaI takes place similarly to the synthesis of oligonucleotides in solutionor, preferably, on solid phase, where appropriate with the assistance ofan automatic synthesizer. The oligomer of the formula I can be assembledstepwise by successive condensation of one PNA unit or DNA unit with ineach case one nucleotide base onto an appropriately derivatized supportor onto a growing oligomer chain. However, the assembly can also takeplace in fragment fashion, in which case the fragments are firstsynthesized as polyamide or oligonucleotide structures which are thenlinked to give the polyamide-oligonucleotide of the formula I. However,it is also possible to use building blocks composed of PNA andnucleotide preferably diners, which are then assembled by the methods ofnucleotide chemistry or peptide chemistry to givepolyamide-oligonucleotide derivatives.

The assembly of the oligonucleotide part takes place by processes knownto the skilled worker, such as the triester method, the H-phosphonatemethod or phosphoramidite method, preferably by the standardphosphoramidite chemistry of Caruthers (M. D. Matteucci and M. H.Caruthers, J. Am. Chem. Soc. 103, 3185 (1981)). The polyamide part canbe synthesized by the methods of peptide chemistry known to the skilledworker. If the oligonucleotide part and polyamide part are notseparately synthesized and subsequently linked, the processes used toassemble the oligonucleotide structure and polyamide structure must bemutually compatible, in which connection a preferred embodiment of thesynthesis of the polyamide part is described in the simultaneously filedapplication with the title “PNA synthesis using an amino protectivegroup which is labile to weak acids” (HOE 94/F 060, DE-P 44 08 531.1).

Depending on whether q, r, s and t are 1 or zero, the synthesis startswith the oligonucleotide part or with the polyamide part. The synthesisof compounds of the formula I whose oligonucleotide part is modified atthe 3′ and/or at the 5′ end takes place in respect of thesemodifications by the processes described in EP-A 0 552 766 (HOE 92/F012) (compare synthetic scheme for DNA). The synthesis of compounds ofthe formula I takes place in respect of the polyamide part by theprocess described in the simultaneously filed application with the title“PNA synthesis using an amino protective group which is labile to weakacids (HOE 94/F 060, DE-P 44 08 531.1) (compare synthetic scheme forPNA).

Synthetic scheme for DNA

-   -   [anchor group]-[polymer]

-   1. ↓ coupling on of PG-(Nu′)-active PG-(Nu′)-[anchor    group]-[polymer]

-   2. ↓ elimination of protective group PG H-(Nu′)-[anchor    group]-[polymer]

-   3. ↓ repetition of steps 1 and 2 (n−1) times H-(Nu′)_(n)-[anchor    group]-[polymer]

-   4. ↓ coupling on of R⁰-V-active R⁰-V-(Nu′)_(n)-[anchor    group]-[polymer]

-   5. ↓ elimination of polymer and protective groups R⁰-V-(Nu)_(n)

Synthetic scheme for PNA

-   -   [anchor group]-[polymer]

-   1. ↓ coupling on of PG-(Q′)-OH PG-(Q′)-[anchor group]-[polymer]

-   2. ↓ elimination of protective group PG H-(Q′)-[anchor    group]-[polymer]

-   3. ↓ repetition of steps 1 and 2 (1-1) times H-(Q′)_(l)-[anchor    group]-[polymer]

-   4. ↓ coupling on of PG-[B′/X]—OH PG-[B′/X]-(Q′)_(l)-[anchor    group]-[polymer]

-   5. ↓ elimination of protective group PG H-[B′/X]-(Q′)_(l)-[anchor    group]-[polymer]

-   6. ↓ repetition of steps 4 and 5 (n-1) times    H-[B′/X]_(n)-(Q′)_(l)-[anchor group]-[polymer]

-   7. ↓ coupling on of PG-(A′)-OH PG-(A′)-[B′/X]_(n)-(Q′)_(l)-[anchor    group]-[polymer]

-   8. ↓ elimination of protective group PG    H-(A′)-[B′/X]_(n)-(Q′)_(l)-[anchor group]-[polymer]

-   9. ↓ repetition of steps 7 and 8 (k-1) times    H-(A′)_(k)-[B′/X]_(n)-(Q′)_(l)-[anchor group]-[polymer]

-   10. ↓ coupling on of the group R⁰    R⁰-(A′)_(k)-[B′/X]_(n)-(Q′)_(l)-[anchor group]-[polymer]

-   11. ↓ elimination of polymer and protective groups    R⁰-(A)_(k)-[B/X]_(n)—(Q)_(l)-Q^(o)

The meanings in this are:

-   PG protective group, preferably a protective group labile to weak    acid;-   Nu′ nucleotide unit whose exocyclic amino group is protected by a    suitable protective group;-   Nu′-active an activated derivative customary in nucleotide    chemistry, such as, for example, of a phosphoramidite, a    phosphodiester or an H-phosphonate;-   A′, B′ and Q′ are the forms of A, B and Q which are protected where    appropriate.

Synthetic scheme for PNA/DNA hybrids of the formula IF[(DNA-Li)_(q)(PNA-Li)_(r)(DNA-Li)_(s)(PNA)_(t)]_(x)F′  (I)

For q=r=s=t=1 and x=1, the following outline of the synthesis applies:

-   1. ↓ synthesis of the end group F′; where appropriate conjugation to    polymer PG-F′-   2. ↓ elimination of protective group PG H—F′-   3. ↓ conjugation of the polyamide structure PNA-F′-   4. ↓ coupling on of a linker Li-PNA-F′-   5. ↓ conjugation of the nucleotide structure DNA-Li-PNA-F′-   6. ↓ coupling on of a linker Li-DNA-Li-PNA-F′-   7. ↓ repetition of steps 3 to 5 DNA-Li-PNA-Li-DNA-Li-PNA-F′-   8. ↓ coupling on of the end group F F-DNA-Li-PNA-Li-DNA-Li-PNA-F′

The coupling on of the linker building block can be omitted ifappropriate junctions are present in the PNA or DNA building blocks.

For clarification, a synthetic scheme for PNA/DNA hybrids of the formulaI is shown and explains by way of example the preparation of a hybridoligomer in which q=r=s=t=1 and x=1. Initially, the end group F issynthesized by known processes and, in the case of solid-phasesynthesis, coupled to a polymeric support (step 1). After elimination ofthe protective group PG (step 2), which preferably takes place in weaklyacidic medium, the polyamide building blocks are coupled on to thedesired length of the PNA part (step 3). As junction to the DNA part itis now possible to attach a linker unit (step 4). The conjugation of thenucleotide structure then takes place by successive condensation on ofthe nucleotide building blocks (step 5), preferably by the knownphosphoramidite method. After a linker which makes it possible to joinDNA to PNA has been condensed on (step 6), in turn a polyamide structureis assembled. Introduction of a linker which makes it possible to joinPNA to DNA, conjugation of another DNA structure (step 7) and finalcoupling on of the end group F (step 8) result in the hybrid molecule[F-DNA-Li-PNA-Li-DNA-Li-PNA-F′]. The linker building blocks can in thiscase also contain nucleotide bases. To synthesize a hybridF-DNA-Li-PNA-Li-F′ (q=r=1, s=t=zero), for example first steps 1-5 arecarried out and then the synthesis is completed with step 8.

To synthesize a hybrid F-PNA-Li-DNA-F′ (r=s=1, q=t=zero), for examplefirst steps 1-2 are carried out, then steps 5-6 follow, followed by step3 and completion of the synthesis with step 8.

To synthesize a hybrid F-PNA-Li-DNA-Li-PNA-F′ (r=s=t=1, q=zero), thesynthesis starts with steps 1-6. After repetition of step 3, thesynthesis is completed with step 8.

If x in formula I is >1, then steps 2-7 must be repeated whereappropriate. After assembly of the polymeric chains, the PNA/DNA hybridsmust in the case of solid-phase synthesis be cleaved off the supportand, where appropriate, the protective groups on the bases, aminoacidside chains and end groups must be eliminated.

However, the PNA part and DNA part can also be synthesized separately byknown methods and subsequently coupled together via appropriateactivation of at least one component. Activation of the PNA partpreferably takes place via the carboxylic acid group, for example asactive ester or isothiocyanate, which are then reacted with reactivegroups in the DNA part, preferably an amino group. Activation of the DNApart takes place, for example, in the form of a cyanogen bromidecondensation known per se, in which the activated phosphatefunctionality is reacted with a reactive group in the PNA part,preferably an amino group.

It has been found, surprisingly, that the oligomers of the formula Iaand Ib have a greatly increased cellular uptake by comparison with purePNAs. This improved cellular uptake is very crucial because antisense-or triplex-forming oligomers are able to act only if they areefficiently taken up by cells. Their hybridization behavior is likewisemore favorable than in the case of pure PNAS because they preferentiallylead to antiparallel duplex formation. Compared with normaloligonucleotides, they have an improved nuclease stability, which isexpressed by an increased biological activity. The binding affinity tocomplementary nucleic acids is better than the other nuclease-stableoligonucleotides such as, for example, phosphorothioates ormethylphosphonates. The binding affinity of the compounds according tothe invention is at least equally good, but usually better, bycomparison with natural oligonucleotides, which are rapidly degradedunder serum conditions. The increase in the binding affinity depends onthe length of the PNA part. Pure PNAs showed a potent cytotoxic effectat concentrations >5 μM in cell-culture experiments, whereas thecompounds according to the invention did not damage the cells. It hasfurthermore been found that compounds of the formula I inhibit,depending on the base sequence of the PNA part and DNA part, theexpression of specific genes, for example of enzymes, receptors orgrowth factors, in cell culture and in selected examples in animalmodels.

Further advantages of the PNA/DNA oligomers and PNA/RNA oligomerscomprise the possibility of stimulating cellular endonucleases such as,for example, RNase H and RNase L. In contrast to PNAs, the PNA-DNAchimeras according to the invention which have some deoxyribonucleotideunits are able, after binding to the complementary target RNA, to cleavethe latter in a sequence-specific manner owing to induction of cellularRNase H. A particular embodiment of the oligomers according to theinvention furthermore comprises those which are composed of PNA part anda 2′,5′-linked oligoadenylate part, preferably tetraadenylate or itscordycepin analog, and which activate cellular RNase L.

The present invention extends very generally to the use of compounds ofthe formula I as therapeutically active ingredients of a pharmaceutical.By therapeutically active polyamide-oligonucleotide derivatives is meantin general antisense oligonucleotides, triple helix-formingoligonucleotides, aptamers or ribozymes, especially antisenseoligonucleotides.

The pharmaceuticals of the present invention can be used, for example,to treat diseases caused by viruses, for example by HIV, HSV-1, HSV-2,influenza, VSV, hepatitis B or papilloma viruses.

Antisense polyamide-oligonucleotide derivatives according to theinvention which are active against such targets have, for example, thefollowing base sequence. The length and position of the PNA part and DNApart in these sequences can be altered appropriately to achieve optimalproperties.

-   a) against HIV, for example

5′-A C A C C C A A T T C T G A A A A T (I) G G-3′ or 5′-A G G T C C C TG T T C G G G C G (II) C C A-3′ or 5′-G T C G A C A C C C A A T T C T GA A A A (III) T G G A T A A-3′ or 5′-G C T A T G T C G A C A C C C A A TT C T (IV) G A A A-3′ or 5′-T C G T C G C T G T C T C C G C T T C T T(VI) C T T C C T G C C A or

-   b) against HSV-1, for example

5′-G C G G G G C T C C A T G G G G G (VII) T C G-3′

The pharmaceuticals of the present invention are also suitable, forexample, for the treatment of cancer. In this connection, it is possibleto use, for example, polyamide-oligonucleotide sequences which aredirected against targets which are responsible for the development ofcancer or the growth of cancers. Examples of such targets are:

-   1) nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb,    c-fos, c-fos/jun, PCNA, p120-   2) cytoplasmic/membrane-associated oncoproteins such as, for    example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos,    c-src, c-abl-   3) cellular receptors such as, for example, the EGF receptor,    c-erbA, retinoid receptors, protein kinase regulatory subunit, c-fms-   4) cytokines, growth factors, extracellular matrix such as, for    example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, bFGF, myeloblastin,    fibronectin.

Antisense polyamide-oligonucleotides of the formula I according to theinvention which are active against such targets have, for example, thefollowing base sequence:

-   a) against c-Ha-ras, for example

5′-C A G C T G C A A C C C A G C-3′ (VIII)

-   c) c-myc, for example

5′-G G C T G C T G G A G C G G G G C A (IX) C A C-3′ 5′-A A C G T T G AG G G G C A T-3′ (X)

-   d) c-myb, for example

5′-G T G C C G G G G T C T T C G G G C-3′ (XI)

-   e) c-fos, for example

5′-G G A G A A C A T C A T G G T C G A (XII) A A G-3′ 5′-C C C G A G A AC A T C A T G G T C G (XIII) A A G-3′ 5′-G G G G A A A G C C C G G C A AG (XIV) G G G-3′

-   f) p120, for example

5′-C A C C C G C C T T G G C C T C C C A C-3′ (XV)

-   g) EGF receptor, for example

5′-G G G A C T C C G G C G C A G C G C-3′ (XVI) 5′-G G C A A A C T T T CT T T T C C (XVII) T C C-3′

-   h) p53 tumor suppressor, for example

5′-G G G A A G G A G G A G G A T G (XVIII) A G G-3′ 5′-G G C A G T C A TC C A G C T T C G (XIX) G A G-3′r

The pharmaceuticals of the present invention are furthermore suitable,for example, for the treatment of diseases which are influenced byintegrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2,ICAM, VCAM or ELAM.

Antisense polyamide-oligonucleotide derivatives according to theinvention which are active against such targets have, for example, thefollowing base sequence:

-   a) VLA-4, for example

5′-G C A G T A A G C A T C C A T A T C-3′ (XX) or

-   b) ICAM, for example

5′-C C C C C A C C A C T T C C C C T C T (XXI) C-3′ 5′-C T C C C C C A CC A C T T C C C C T (XXII) C-3′ 5′-G C T G G G A G C C A T A G C G A G(XXIII) G-3′

-   c) ELAM-1, for example

5′-A C T G C T G C C T C T T G T C T C A G (XXIV) G-3′ 5′-C A A T C A AT G A C T T C A A G A G T T (XXV) C-3′

The pharmaceuticals of the present invention are also suitable, forexample, for preventing restenosis. In this connection, examples ofpolyamide-oligonucleotide sequences which can be used are those directedagainst targets which are responsible for proliferation or migration.Examples of such targets are:

-   1) nuclear transactivator proteins and cyclins such as, for example,    c-myc, c-myb, c-fos, c-fos/jun, cyclins and cdc2 kinase-   2) mitogens or growth factors such as, for example, PDGF, bFGF, EGF,    HB-EGF and TGF-β-   3) cellular receptors such as, for example, bFGF receptor, EGF    receptor and PDGF receptor.

Antisense polyamide-oligonucleotides according to the invention of theformula I which are active against such targets have, for example, thefollowing base sequence:

-   a) c-myb

5′-G T G T C G G G G T C T C C G G G C-3′ (XXVI)

-   b) c-myc

5′-C A C G T T G A G G G G C A T-3′ (XXVII)

-   c) cdc2 kinase

5′-G T C T T C C A T A G T T A C T C A-3′ (XXVIII)

-   d) PCNA (proliferating cell nuclear antigen of rat)

5′-G A T C A G G C G T G C C T C A A A-3′ (XXIX)

The pharmaceuticals can be used, for example, in the form ofpharmaceutical products which can be administered orally, for example inthe form of tablets, coated tablets, hard or soft gelatin capsules,solutions, emulsions or suspensions. Inclusion of the pharmaceuticals inliposomes, which optionally contain further components such as proteins,is a likewise suitable administration form. They can also beadministered rectally, for example in the form of suppositories, orparenterally, for example in the form of injection solutions. To producepharmaceutical products, these compounds can be processed intherapeutically inert organic and inorganic excipients. Examples of suchexcipients for tablets, coated tablets and hard gelatin capsules arelactose, corn starch or derivatives thereof, talc and stearic acid orsalts thereof. Suitable excipients for producing solutions are water,polyols, sucrose, invert sugar and glucose. Suitable excipients forinjection solutions are water, alcohols, polyols, glycerol and vegetableoils. Suitable excipients for suppositories are vegetable and hardenedoils, waxes, fats and semiliquid polyols. The pharmaceutical productsmay also contain preservatives, solvents, stabilizers, wetting agents,emulsifiers, sweeteners, colorants, flavorings, salts to alter theosmotic pressure, buffers, coating agents, antioxidants and, whereappropriate, other therapeutic active substances.

Preferred administration forms are topical applications, localapplications such as, for example, with the aid of a catheter or elseinjections. For injection, the antisense polyamide-oligonucleotidederivatives are formulated in a liquid solution, preferably in aphysiologically acceptable buffer such as, for example, Hank's solutionor Ringer's solution. The antisense polyamideoligonucleotides can,however, also be formulated in solid form and be dissolved or suspendedbefore use. The dosages preferred for systemic administration are about0.01 mg/kg to about 50 mg/kg of body weight and day.

The invention extends very generally to the use of compounds of theformula I as DNA probes or primers in DNA diagnosis, in particular inthe sense of the gene probes mentioned in HOE 92/F 406 (EP-A 0 602 524),and generally as aids in molecular biology.

Gene probes, also called DNA probes or hybridization probes, play alarge part in DNA diagnosis for sequence-specific detection ofparticular genes. A gene probe is generally composed of a recognitionsequence and of a suitable labeling group (label). The specificity ofthe determination of a target sequence in an analytical sample byhybridization with a complementary gene probe is determined by therecognition sequence and its chemical structure. The PNAs have theadvantage, compared with oligonucleotides of natural structure, thatthey have a higher affinity for the target sequence. However, thespecificity of the hybridization is reduced because PNAs, in contrast tonatural DNA, are able to bind both in parallel and in antiparallelorientation to single-stranded nucleic acids. The PNA/DNA oligomersaccording to the invention likewise show an increased binding affinitybut very preferentially bind in the desired antiparallel orientation.

It is moreover possible, by appropriate selection of the PNA part andDNA part in a gene probe, to have a beneficial effect on thedifferentiation capacity because base mispairing in the PNA part leadsto a greater depression of the melting temperature of a hybrid than doesa base mispairing in the DNA part. This is particularly important withregard to differentiation in the case of point mutations as occur, forexample, in the transition from protooncogenes into the correspondingoncogenes (pathogenic state). The advantage of the better discriminationbetween pathogenic and non-pathogenic state can also be utilized in theform of the primer property of the PNA/DNA oligomers according to theinvention as long as these have a free 3′-hydroxyl group in the DNApart. PNAs as such have no primer function for polymerases. It has beenfound, surprisingly, that even one nucleoside unit at the end of aPNA/DNA oligomer is sufficient to initiate the DNA polymerase reaction,for example with DNA polymerase (Klenow fragment). Various polymerasescan be employed depending on the characteristics of the PNA/DNA primerand the nature of the template onto which the primer hybridizes in asequence-specific manner. These polymerases are generally commerciallyavailable, such as, for example, Taq polymerase, Klenow polymerase orreverse transcriptase.

Another advantage by comparison with the use of natural oligonucleotideprimers is that the nucleic acid strand which is copied with the aid ofthe PNA/DNA primer and which contains the PNA part at the 5′ end isstable to 5′-exonucleases. It is thus possible to degrade all naturalDNA or RNA sequences in the reaction mixture by 5′-exonucleases withoutattack on the PNA-containing strand.

Another advantage of the PNA/DNA oligomers is that they can also be usedto carry out other biochemical reactions on the DNA part which areimpossible with PNAs themselves. Examples of such reactions are the3′-tailing with 3′-terminal transferase, the digestion with restrictionenzymes in the DNA double-stranded region, and ligase reactions. Forexample, a (PNA)-(DNA)-OH oligomer with free 3′-hydroxyl group can belinked to a second p-(DNA)-(PNA) oligomer which contains a nucleoside5′-phosphate at the 5′ end after hybridization to a complementary DNAauxiliary sequence of natural origin in the presence of a DNA ligase.

(DNA)-(PNA)-(DNA) oligomers can furthermore be incorporated into genes,which is not at present possible with PNAs.

The linkage of labeling groups onto PNA/DNA oligomers takes place bymethods known per se, as described for oligonucleotides or peptides. Thenature of the labeling group can vary within wide limits and dependsessentially on the type of assay used. Known embodiments of gene probeassays are the hybridization protection assay, the energy transfer assayand the kissing probes assay.

PNA/DNA oligomers are additionally particularly suitable for a stranddisplacement assay. In many cases it is advantageous to remove thehybrid which is formed from excess gene probe with the aid of magneticparticles. The stability of the PNA/DNA gene probes according to theinvention is higher than that of conventional DNA probes.

Polymerase chain reaction (PCR) and ligase chain reaction (LCR) aretechniques for target amplification in which the oligomers according tothe invention can likewise be used as primers. The PNA/DNA oligomers canbe used particularly advantageously as gene probes on the Christmas treeprinciple because in this case the PNA/DNA probes can be shorter thancorresponding DNA probes.

EXAMPLES

The abbreviations used for amino acids correspond to the three-lettercode customary in peptide chemistry, as described in Europ. J. Biochem.138, 9 (1984). Other abbreviations used are listed below.

-   Aeg N-(2-Aminoethyl)glycyl, —NH—CH₂—CH₂—NH—CH₂—CO—-   Aeg(a^(MeOBz))    N-(2-Aminoethyl)-N—(N⁶-(4-methoxybenzoyl)-9-adenosylacetyl)-glycyl-   Aeg(c^(Bz)) N-(2-Aminoethyl)-N-(N-4-benzoyl-1-cytosylacetyl)-glycyl-   Aeg(c^(MeOBz))    N-(2-Aminoethyl)-N—(N⁴-(4-methoxybenzoyl)-1-cytosylacetyl)-glycyl-   Aeg(c^(tBuBz))    N-(2-Aminoethyl)-N—(N⁴-(4-tert.butylbenzoyl)-1-cytosylacetyl)-glycyl-   Aeg(g^(iBu))    N-(2-Aminoethyl)-N-(N-2-isobutanoyl-9-guanosylacetyl)-glycyl-   Aeg(g^(2-Ac,4-Dpc))    N-(2-Aminoethyl)-N—(N²-acetyl-O⁴-diphenylcarbamoyl-9-guanosyl)glycyl-   Aeg(t) N-(2-Aminoethyl)-N-((1-thyminyl)acetyl)-glycyl-   Bnpeoc 2,2-[bis(4-Nitrophenyl)]-ethoxycarbonyl)-   Boc tert.-butyloxycarbonyl-   BOI 2-(Benzotriazol-1-yloxy)-1,3-dimethylimidazolidinium    hexafluorophosphate-   BOP Benzotriazolyl-1-oxy-tris(dimethylamino)phosphonium    hexafluorophosphate-   BroP Bromo-tris(dimethylamino)phosphonium hexafluorophosphate-   BSA N,O-bis(Trimethylsilyl)-acetamide-   But tert.-butyl-   Bz Benzoyl-   Bzl Benzyl-   Cl-Z 4-Chloro-benzyloxycarbonyl-   CPG Controlled pore glass-   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene-   DCM Dichloromethane-   Ddz 2-(3,5-Dimethoxyphenyl)-2-propyloxycarbonyl-   DMF Dimethylformamide-   Dmt di-(4-Methoxyphenyl)phenylmethyl-   Dnpeoc 2-(2,4-Dinitrophenyl)-ethoxycarbonyl-   Dpc Diphenylcarbamoyl-   FAM Fluorescein residue-   Fm 9-Fluorenylmethyl-   Fmoc 9-Fluorenylmethyloxycarbonyl-   H-Aeg-OH N-(2-Aminoethyl)glycine-   HAPyU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium    hexafluorophosphate-   HATU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HBTU O-(Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   HOBt 1-Hydroxybenzotriazole-   HONSu N-Hydroxysuccinimide-   HOObt 3-Hydroxy-4-oxo-3,4-dihydrobenzotriazine-   iBu Isobutanoyl-   MeOBz 4-Methoxybenzoyl-   Mmt 4-Methoxytriphenylmethyl-   Moz 4-Methoxybenzyloxycarbonyl-   MSNT 2-Mesitylenesulfonyl-3-nitro-1,2,4-triazole-   Mtt (4-Methylphenyl)diphenylmethyl-   NBA Nitrobenzyl alcohol-   NMP N-Methylpyrrolidine-   Obg N-(4-oxybutyl)glycyl, —O—(CH₂)₄—NH—CH₂—CO—-   Obg(t) N-(4-oxybutyl)-N-((1-thyminyl)acetyl)-glycyl-   Oeg N-(2-oxyethyl)glycyl, —O—CH₂—CH₂—NH—CH₂—CO—-   Oeg(t) N-(2-Oxyethyl)-N-((1-thyminyl)acetyl)-glycyl-   Opeg N-(5-Oxypentyl)glycyl, —O—(CH₂)₅—NH—CH₂—CO—-   Opeg(t) N-(5-Oxypentyl)-N-((1-thyminyl)acetyl)-glycyl-   Oprg N-(3-Oxypropyl)glycyl, —O—(CH₂)₃—NH—CH₂—CO—-   Oprg(t) N-(3-Oxypropyl)-N-((1-thyminyl)acetyl)-glycyl-   Pixyl 9-(9-Phenyl)xanthenyl-   PyBOP Benzotriazolyl-1-oxytripyrrolidinophosphonium    hexafluorophosphate-   PyBroP Bromotripyrrolidinophosphonium hexafluorophosphate-   TAPipU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-bis (pentamethylene)    uronium tetrafluoroborate-   TBTU O-(Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    tetrafluoroborate-   tBu tert.-Butyl-   tBuBz 4-tert.Butylbenzoyl-   TDBTU    O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium    tetrafluoroborate-   TDO 2,5-Diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide-   Teg N-(2-Thioethyl)glycyl, —S—CH₂—CH₂—NH—CH₂—CO—-   Teg(t) N-(2-Thioethyl)-N-((1-thyminyl)acetyl)-glycyl-   TFA Trifluoroacetic acid-   THF Tetrahydrofuran-   TNTU O-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium    tetrafluoroborate-   TOTU    O-[(Cyano(ethoxycarbonyl)methylene)amino]-1,1,3,3-tetramethyluronium    tetrafluoroborate-   TPTU O-(1,2-Dihydro-2-oxo-1-pyridyl)-1,1,3,3-tetramethyluronium    tetrafluoroborate-   Trt Trityl-   TSTU O—(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate-   Z Benzyloxycarbonyl-   MS(ES⁺) Electrospray mass spectrum (positive ion)-   MS(ES⁻) Electrospray mass spectrum (negative ion)-   MS(DCI) Desorption chemical ionization mass spectrum-   MS(FAB) Past atom bombardment mass spectrum

Example 1 1-Hydroxy-6-((4-methoxyphenyl)-diphenylmethylamino)hexane

Mmt-hex

6-Amino-1-hexanol (1 g; 8.55 mmol) is dissolved in anhydrous pyridine (7ml), and triethylamine (0.2 ml) is added. To this solution is added overthe course of 45 minutes a solution of (4-methoxyphenyl)diphenylmethylchloride (2.5 g; 8.12 mmol) in anhydrous pyridine (9 ml). The reactionsolution is stirred further at 22° C. for 30 minutes and stopped byadding methanol (3 ml). The solution is concentrated in a rotaryevaporator, and the resulting residue is coevaporated with toluene threetimes to remove the pyridine. The resulting residue is dissolved inethyl acetate, and this solution is washed successively with a saturatedsodium bicarbonate solution, with water and with a saturated potassiumchloride solution. The organic phase is dried (Na₂SO₄), filtered andconcentrated in vacuo. The crude product is purified by silica gelchromatography using heptane:ethyl acetate: triethylamine/49.5:49.5:1.

Yield: 1.64 g

MS (FAB,NBA/LiCl) 396.3 (M+Li)⁺, 390.3 (M+H)⁺, 273.2 (Mmt)⁺

R_(f) 0.44 (heptane:ethyl acetate=1:1)

Example 2 6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexyl hemisuccinate

Mmt-hex-succ

1-Hydroxy-6-((4-methoxyphenyl) diphenylmethylamino) hexane (1.00 g; 2.57mmol) is dissolved in anhydrous pyridine (10 ml). To this solution areadded succinic anhydride (0.257 g; 2.57 mmol) and4-dimethylaminopyridine (31.3 mg; 0.257 mmol). After stirring at 22° C.for 3 hours, further succinic anhydride (25.7 mg; 0.257 mmol) and4-dimethylaminopyridine (62.6 mg; 0.56 mmol) are added, and thissolution is heated at 50° C. for 6 hours. After a further 16 hours at22° C., the mixture is concentrated, the residue is taken up in ethylacetate, and the resulting solution is washed with ice-cold 5% strengthaqueous citric acid. After the org. phase has been dried (Na₂SO₄), thesolution is concentrated in a rotary evaporator. Purification of theresidue by silica gel chromatography using 50% CH₂Cl₂/1% triethylaminein ethyl acetate and then using 5% methanol/1% triethylamine indichloromethane affords the required compound as colorless oil.

MS (ES⁻) 978.0 (2M−H)⁻, 488.3 (M−H)⁻

R_(f) 0.30 (CH₂Cl₂:ethyl acetate=1:1).

Example 3 6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexylsuccinylamido-Tentagel

(Mmt-hex-succ-Tentagel)

The amino form of Tentagel^(R) (Rapp polymers) (0.5 g; 0.11 mmol aminogroups) is left to swell in 4-ethylmorpholine (0.1 ml) and DMF (5 ml)for 10 minutes. A solution of6-((4-methoxyphenyl)diphenylmethylamino)-1-hexyl hemisuccinate (97.4 mg;0.165 mmol), 4-ethylmorpholine (15.9 mg; 0.138 mmol; 17.4 ml) and TBTU(52.9 mg; 0.165 mmol) in DMF (3 ml) is then added, and the suspension isshaken at 22° C. for 16 hours. The derivatized Tentagel support isfiltered off and washed successively with DMF (3×3 ml), CH₂C1₂ (3×1 ml)and diethyl ether (3×1 ml) and dried. Unreacted amino groups are blockedby treatment with acetic anhydride/lutidine/1-methylimidazole in THF (1ml) for 1 hour. The completed support is washed with CH₂Cl₂ (3×1 ml) anddiethyl ether (3×1 ml) and dried in vacuo. Based on themonomethoxytrityl group introduced, the loading is 168 mmolg⁻¹.

Example 4 6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexylsuccinylamidopropyl-controlled pore glass

(Mmt-hex-succ-CPG)

The preparation takes place in analogy to the description in Example 3starting from aminopropyl-CPG (supplied by Fluka) (550 Angstrom; 1.0 g)and 6-((4-methoxyphenyl)diphenylmethylamino)-1-hexyl hemisuccinate (48.7mg; 0.082 mmol), 4-ethylmorpholine (7.6 ml) and TBTU (26.4 mg; 0.082mmol) in DMF (3 ml). The loading of the Mmt-hex-succCPG is 91 mmolg⁻¹.

Example 5N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N—(N⁴-(4-tert-butylbenzoyl)-1-cytosylacetyl)glycine

(Mmt-Aeg(c^(tBuBz))-OH)

1.63 g (2.28 mmol) ofN-((4-methoxyphenyl)diphenylmethylamino)ethyl-N—(N⁴-(4-tert-butylbenzoyl)-1-cytosylacetyl)glycinemethyl ester were dissolved in a mixture of 10 ml of dioxane and 1 ml ofwater and, while stirring at 0° C., 4.56 ml of 1 N NaOH were addeddropwise. After 2 h, the pH was adjusted to 5 by dropwise addition of 1N KHSO₄, and precipitated salts were filtered off and washed with alittle dioxane. The combined filtrates were evaporated in vacuo, and theresidue was coevaporated twice with methanol and dichloromethane. Thecrude product obtained in this way was purified by chromatography onsilica gel using a gradient of 2-10% methanol and 1% triethylamine indichloromethane. The fractions containing the product were combined andconcentrated in vacuo. Excess triethylamine still present was removed bycoevaporation with pyridine and then toluene. 0.831 g of product wasobtained as an almost white foam.

Electrospray MS (negative ion) 700.7 (M−H)⁻.

R_(f) 0.28 (CH₂Cl₂:MeOH/9:1), 0.63 (CH₂Cl₂:MeOH/7:3).

Example 6N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N-((1-thyminyl)acetyl)glycine

(Mmt-Aeg(t))-OH

The product from the above reaction was dissolved in a mixture of 10 mlof dioxane and 2 ml of water, the solution was cooled to 0° C., and 1 Nsodium hydroxide solution was added dropwise until the pH reached 11.After a reaction time of 2 h, the reaction was complete and the solutionwas adjusted to pH 5 by cautious addition of 2 N KHSO₄ solution. Thesolution was extracted three times with ethyl acetate, and the combinedorganic phases were dried over sodium sulfate and concentrated in vacuo.The crude product obtained in this way was purified by chromatography onsilica gel using a gradient of 5-10% methanol and 1% triethylamine indichloromethane. The fractions containing the product were combined andconcentrated in vacuo. Excess triethylamine still present was removed bycoevaporation with pyridine and then toluene. 1.065 g of product wereobtained as a colorless foam.

Electrospray MS (negative ion) 1112.0 (2M−H)⁻, 555.3 (M−H)⁻

R_(f) 0.28 (CH₂Cl₂:MeOH/8:2).

Example 7N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N—(N²-isobutanoyl-9-guanosylacetyl)glycine

(Mmt-Aeg(g^(iBu))-OH

N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N—(N²-isobutanoyl-9-guanosylacetyl)glycinemethyl ester (1.15 g; 1.72 mmol) is dissolved in dioxane (10 ml) and, at0° C., 1 M aqueous sodium hydroxide solution (10.32 ml) is addeddropwise in 5 portions over a period of 2.5 h. After a further reactiontime of 2 h at room temperature, the solution is adjusted to pH 5 bydropwise addition of 2 M aqueous potassium bisulfate solution. Theprecipitated salts are filtered off and washed with a little dioxane.The combined filtrates are evaporated to dryness in vacuo, and theresidue is coevaporated twice each with ethanol and 1/1dichloromethane:methanol. Purification takes place by columnchromatography on silica gel by elution with a gradient of 10-20%methanol in dichloromethane (with 1% triethylamine). The product isobtained as a white foam.

Yield: 1.229 g

ESMS (negative ion): 650.3 (M−H)⁻

R_(f) 0.25 (dichloromethane:methanol/8:2)

Example 8 N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N—(N-⁶-(4-methoxybenzoyl)-9-adenosylacetyl)glycine

(Mmt-Aeg(a^(MeOBz))-OH)

N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N—(N⁶-(4-methoxybenzoyl)-9-adenosylacetyl)glycinemethyl ester (1.70 g; 2.38 mmol) is dissolved in dioxane (10 ml) and, at0° C., 1 M aqueous sodium hydroxide solution (10.32 ml) is addeddropwise in 5 portions over a period of 2.5 h. After a further reactiontime of 2 h at room temperature, the solution is adjusted to pH 5 bydropwise addition of 2 M aqueous potassium bisulfate solution. Theprecipitated salts are filtered off and washed with a little dioxane.The combined filtrates are evaporated to dryness in vacuo, and theresidue is coevaporated twice each with ethanol and 1/1dichloromethane:methanol. Purification takes place by columnchromatography on silica gel by elution with a gradient of 10-20%methanol in dichloromethane (with 1% triethylamine). The product isobtained as a white foam.

Yield: 1.619 g

ESMS (negative ion): 698.3 (M−H)⁻

R_(f) 0.10 (dichloromethane:methanol/8:2)

Example 9N-((4-Methoxyphenyl)diphenylmethyloxy)ethyl-N-((1-thyminyl)acetyl)glycine

(Mmt-Oeg(t)-OH)

0.5 g (1.28 mmol) of N-((4-methoxyphenyl)diphenylmethyloxy)ethylglycinewas suspended in 10 ml of DMF, and 0.47 ml (1.92 mmol) of BSA was addeddropwise. Then, 0.7 ml (5.1 mmol) of triethylamine and 0.26 g (1.28mmol) of chlorocarboxymethylthymine were successively added. Thereaction mixture was stirred at room temperature for 4 h and then afurther 65 mg (0.32 mmol) of chlorocarboxymethylthymine were added, andthe mixture was stirred for 16 h. The solvent was then stripped off invacuo, and the crude product was purified on a silica gel column using agradient of 5-15% methanol and 1% triethylamine in dichloromethane. Thefractions containing the product were combined and concentrated invacuo. The resulting brownish oil was dissolved in a littledichloromethane, and the product was precipitated by adding diethylether. The product was obtained as an almost white powder.

Yield: 0.219 g

Electrospray MS (negative ion) 556.3 (M-H)⁻.

R_(f) 0.54 (CH₂Cl₂:MeOH/8:2).

Example 10 4-Nitrophenyl 4-(4,4′-dimethoxytrityloxy)butyrate

Dmt-but-NPE

The sodium salt of 4-hydroxybutyric acid (1.26 g; 10 mmol) is dissolvedin anhydrous pyridine (30 ml), and 4,4′-dimethoxytrityl chloride (3.39g; 3.05 mmol) is added. After 16 hours, 4-nitrophenol (1.39 g; 10 mmol)and N,N′-dicyclohexylcarbodiimide (2.06 g; 10 mmol) are added, and themixture is stirred at 22° C. for a further 48 hours. The precipitateddicyclohexylurea is filtered off and washed with dichloromethane. Thefiltrate is concentrated and the resulting residue is coevaporated twicewith toluene. The residue is purified on a silica gel column (10-50%ethyl acetate and 1% triethylamine in petroleum ether). The desiredcompound is obtained in the form of a pale yellowish-colored oil.

Yield: 2.694 g

MS (FAB, MeOH/NBA/LiCl) 534.2 (M+Li)⁺, 527.2 M⁺.

R_(f) 0.34 (petroleum ether:ethyl acetate=75:25)

Example 11 H-Oprg(t)-OH

3.68 g of thyminylacetic acid are dissolved in 20 ml of dry DMF, and6.65 g of TOTU and 2.77 ml of triethylamine are added. The mixture isstirred at room temperature for 30 min and then slowly added dropwise toa solution composed of 5.32 g of (3-hydroxypropyl)glycine, 20 ml ofwater, 20 ml of DMF and 5.54 ml of triethylamine. The mixture is stirredat room temperature for 1 h and then concentrated in a rotary evaporatorin vacuo. The residue is taken up in water, adjusted to pH 1.5 with 1 Nhydrochloric acid and extracted with ethyl acetate. The aqueous phase isadjusted to pH 5 with saturated sodium bicarbonate solution andconcentrated in a rotary evaporator. The residue is mixed with 250 ml ofethanol, and the sodium chloride precipitated thereby is filtered offwith suction. The filtrate is concentrated and the crude product ispurified by chromatography on silica gel usingdichloromethane/methanol/ethyl acetate 10:2:1 with the addition of 1%triethylamine followed by dichloromethane/methanol/ethyl acetate 10:4:1with the addition of 1% triethylamine. The fractions containing theproduct are combined and concentrated in a rotary evaporator in vacuo.

Yield: 3.2 g

R_(f) 0.15 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS(ES⁺): 300.2 (M+H)⁺.

Example 12 Dmt-Oprg(t)-OH

3.2 g of H-Oprg(t)-OH are dissolved in 40 ml of DMF, 5.93 ml oftriethylamine are added and, at 0° C., a solution of 7.25 g of Dmt-Cl in40 ml of dichloromethane is added dropwise over the course of 20 min.The mixture is stirred at room temperature for 2 h, then theprecipitated triethylamine hydrochloride is filtered off, and thefiltrate is concentrated in a rotary evaporator in vacuo. The residue istaken up in dichloromethane and extracted with water, and the organicphase is dried with sodium sulfate and concentrated in a rotaryevaporator in vacuo. The crude product is purified on silica gel usingdichloromethane/methanol/ethyl acetate 10:2:1 with the addition of 1%triethylamine. The fractions containing the product are combined andconcentrated in a rotary evaporator in vacuo.

Yield: 3.46 g

R_(f) 0.28 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS (ES⁺) 602.4 (M+H)⁺.

Example 13 H-Obg(t) OH

2.76 g of thyminylacetic acid are dissolved in 15 ml of dry DMF, and4.92 g of TOTU and 2.08 ml of triethylamine are added. The mixture isstirred at room temperature for 30 min and then slowly added dropwise toa solution composed of 4.41 g of (4-hydroxybutyl)glycine, 10 ml ofwater, 10 ml of DMF and 4.16 ml of triethylamine. The mixture is stirredat room temperature for 3 h and then concentrated in a rotary evaporatorin vacuo. The residue is taken up in water, adjusted to pH 1.5 with 1 Nhydrochloric acid and extracted with ethyl acetate. The aqueous phase isadjusted to pH 5 with saturated sodium bicarbonate solution andconcentrated in a rotary evaporator. The crude product is purified bychromatography on silica gel using dichloromethane/methanol/ethylacetate 10:2:1 with the addition of 1% triethylamine. The fractionscontaining the product are combined and concentrated in a rotaryevaporator in vacuo.

Yield: 3.7 g

R_(f) 0.11 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS (ES⁺) 314.2 (M+H)⁺.

Example 14 Dmt-Obg (t)-OH

3.6 g of H-Obg(t)-OH are dissolved in 40 ml of DMF, 9.5 ml oftriethylamine are added and, at 0° C., a solution of 15.4 g of Dmt-Cl in40 ml of dichloromethane is added dropwise over the course of 15 min.The mixture is stirred at room temperature for 2 h, a further 40 ml ofdichloromethane are added, then the precipitated triethylaminehydrochloride is filtered off, and the filtrate is concentrated in arotary evaporator in vacuo. The residue is taken up in dichloromethaneand extracted with water, and the organic phase is dried with sodiumsulfate and concentrated in a rotary evaporator in vacuo. The crudeproduct is purified on silica gel using dichloromethane/methanol/ethylacetate 15:1:1 with the addition of 1% triethylamine. The fractionscontaining the product are combined and concentrated in a rotaryevaporator in vacuo.

Yield: 3.45 g

R_(f) 0.29 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS (ES⁺+LiCl) 622.3 (M+Li)⁺.

Example 15 H-Opeg(t) OH

2.76 g of thyminylacetic acid are dissolved in 15 ml of dry DMF, and4.92 g of TOTU and 2.08 ml of triethylamine are added. The mixture isstirred at room temperature for 30 min and then slowly added dropwise toa solution composed of 4.83 g of (5-hydroxypentyl)glycine, 10 ml ofwater, 10 ml of DMF and 4.16 ml of triethylamine. The mixture is stirredat room temperature for 3 h and then concentrated in a rotary evaporatorin vacuo. The residue is taken up in water, adjusted to pH 1.5 with 1 Nhydrochloric acid and extracted with ethyl acetate. The aqueous phase isadjusted to pH 5 with saturated sodium bicarbonate solution andconcentrated in a rotary evaporator. The crude product is purified bychromatography on silica gel using dichloromethane/methanol/ethylacetate 10:2:1 with the addition of 1% triethylamine. The fractionscontaining the product are combined and concentrated in a rotaryevaporator in vacuo.

Yield: 3.34 g

R_(f) 0.19 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS (DCl) 328.2 (M+H)⁺.

Example 16 Dmt-Opeg(t)-OH

3.2 g of H-Opeg(t)-OH are dissolved in 40 ml of DMF, 6.77 ml oftriethylamine are added and, at 0° C., a solution of 9.94 g of Dmt-Cl in40 ml of dichloromethane is added dropwise over the course of 15 min.The mixture is stirred at room temperature for 2 h, a further 40 ml ofdichloromethane are added, then the precipitated triethylaminehydrochloride is filtered off, and the filtrate is concentrated in arotary evaporator in vacuo. The residue is taken up in dichloromethaneand extracted with water, and the organic phase is dried with sodiumsulfate and concentrated in a rotary evaporator in vacuo. The crudeproduct is purified on silica gel using dichloromethane/methanol/ethylacetate 15:1:1 with the addition of 1% triethylamine. The fractionscontaining the product are combined and concentrated in a rotaryevaporator in vacuo.

Yield: 3.6 g

R_(f) 0.27 (dichloromethane/methanol/ethyl acetate 10:2:1+1%triethylamine)

MS (ES⁺+LiCl) 636.4 (M+Li)⁺.

Example 17

5′-ATC GTC GTA TT-(but)-agtc-hex

The DNA sequence is indicated in capital letters and the PNA sequence isindicated in small letters (example of the structural type XIIa inscheme 1). The PNAs are synthesized, for example, in an Ecosyn D-300 DNAsynthesizer (from Eppendorf/Biotronik, Maintal) or an ABI 380B DNAsynthesizer (from Applied Biosystems, Weiterstadt). The synthesis of theDNA part is carried out in principle by standard phosphoramiditechemistry and commercially obtainable synthesis cycles. For thesynthesis of the PNA part the methods of peptide synthesis areapproximated to the DNA synthesis cycles as explained hereinafter.

Formula VIII

R⁵ R⁶ R² V VIII a NC—CH₂CH₂—O— —N(i-C₃H₇)₂ H O VIII b CH₃ —N(i-C₃H₇)₂ HO VIII c C₆H₅ —N(i-C₃H₇)₂ H O VIII d C₆H₅—C(O)—S(CH₂)₂—S-N-pyrrolidin-1-yl H O VIII e NC—CH₂CH₂—O— —N(i-C₃H₇)₂ OCH₃ O VIII fNC—CH₂CH₂—O— —N(i-C₃H₇)₂ H NH

with n=1-8, preferably 1-5,

3 μmol of the CPG support loaded with Mmt-hex-succ (loading 91 μmol/g)from Example 4 are treated successively with the following reagents:Synthesis of the PNA Part (agtc-hex):

-   1. dichloromethane-   2. 3% trichloroacetic acid in dichloromethane-   3. acetonitrile abs.-   4. 3.5 M solution of 4-ethylmorpholine in acetonitrile    (neutralization)-   5. 0.4 X solution of (Mmt-Aeg(c^(tBuBz))-OH) from Example 5 in    acetonitrile:DMF=9:1/0.9 M solution of ByBOP in acetonitrile/3.5 M    solution of 4-ethylmorpholine in acetonitrile (coupling time of 10    minutes).-   6. step 5 is repeated four times.-   7. acetonitrile

Steps 1 to 7, called a PNA reaction cycle hereinafter, are repeated 3times to assemble the PNA part, using in step 5 in each case the monomerbuilding block, necessary according to the sequence, from Examples 5 to8.

Conjugation of the linker (agtc-hex--->(but)-agtc-hex):

-   8. repeat steps 1 to 4 from above-   9. 4-nitrophenyl 4-(4,4′-dimethoxytrityloxy)butyrate (105 mg) from    Example 10 and hydroxybenzotriazole (27 mg) in 2 ml of NEM in DMF    for 15 hours-   10. wash with DMF-   11. wash with acetonitrile-   12. dichloromethane    Synthesis of the DNA Part    ((but)-agtc-hex)-->5′-ATC GTC GTA TT-(but)-agtc-hex):-   13. acetonitrile abs.-   14. 3% trichloroacetic acid in dichloromethane-   15. acetonitrile abs.-   16. 10 μmol of β-cyanoethyl 5′-O-dimethoxytritylthymidine    3′-diisopropylphosphoramidite and 50 μmol of tetrazole in 0.3 ml of    acetonitrile abs.-   17. acetonitrile-   18. 20% acetic anhydride in THF with 40% lutidine and 10%    dimethylaminopyridine-   19. acetonitrile-   20. iodine (1.3 g in THF/water/pyridine; 70:20:5=v:v:v)

Steps 13 to 20, called a DNA reaction cycle hereinafter, are repeated 10times to assemble the nucleotide part, using in step 16 in each case theβ-cyanoethyl 5′-O-dimethoxytrityl(nucleotide base)3′-diisopropylphosphoramidite corresponding to the sequence.

After the synthesis is complete, the dimethoxytrityl group is eliminatedas described in steps 1 to 3. The oligomer is cleaved off the supportand, at the same time, the β-cyanoethyl groups are eliminated bytreatment with ammonia for 1.5 hours. To eliminate the exocyclic aminoprotective groups, the ammoniacal solution is kept at 55° C. for 5hours. 180 OD₂₆₀ of the resulting crude product (325 OD₂₆₀) of 5′-ATCGTC GTA TT-(but)-agtc-hex are purified by polyacrylamide gelelectrophoresis. Desalting on a Biogel^(R) column (from Biorad) resultsin 50 OD₂₆₀ of high-purity oligomer from this.

Example 18 5′-ATC GTC GTA TT-(Oeg(t))-agtc-hex

(Example of Structural Type Xa in Scheme 2; see Example 9 forExplanation of Oeg(t))

The synthesis takes place in analogy to the description in Example 17but in step 9 coupling the linker building block Mmt-Oeg(t)-OH fromExample 9, in place of the p-nitrophenyl Dmt-butyrate, under theconditions described in step 5. 135 OD₂₆₀ of the resulting crude product(235 OD₂₆₀) of 5′-ATC GTC GTA TT-(Oeg(t)-agtc-hex are purified bypolyacrylamide gel electrophoresis. Desalting on a Biogel^(R) column(from Biorad) results in 20 OD₂₆₀ of high-purity oligomer from this.

Example 19 N-ggg g(5′NH—C)T C_(S)C_(S)A_(S) TGG GG_(S)G_(S) T (sequencecomplementary to HSV-1)

(Example of Structural Type XI in Scheme 2; _(S) Means aPhosphorothioate Bridge; (5′NH—C) Means a 5′-aminocytidylate Residue; NEquals Amino Terminus)

The synthesis takes place starting from a CPG support on which5′-Dmt-thymidine is bound via its 3′ end. The synthesis of the DNA partis first carried out as described in Example 17 (steps 13 to 20),carrying out the oxidation in step 20 in the case of thephosphorothioate bridges (_(S)) using tetraethylthiuram disulfide (TETD;User Bulletin No. 65 of Applied Biosystems Inc.). A Dmt-protected5′-amino-5′-deoxycytidylate 3′-phosphoramidite building block of theformula VIIIf is used as linker building block. The PNA building blocksare then condensed on in analogy to steps 1 to 7 in Example 17. Afterthe synthesis is complete, the oligomer is cleaved off the support and,at the same time, the β-cyanoethyl groups are eliminated by treatmentwith ammonia for 1.5 hours. To eliminate the exocyclic amino protectivegroups, the ammoniacal solution is kept at 55° C. for 5 hours. Only thenis the monomethoxytrityl group eliminated by treatment with 80% strengthacetic acid at 22° C. for 2 hours. The product is purified bypolyacrylamide gel electrophoresis and desalted on a Biogel^(R) column(from Biorad).

Example 20 5′-G_(Me)G_(Me)G GCT CCA (Oeg(t))gg ggg t-hex

(Example of Structural Type Xa in Scheme 2; _(Me) Means aMethylphosphonate Bridge; see Example 9 for Explanation of Oeg(t))

The synthesis takes place in analogy to the description in Example 18but using the appropriate methylphosphonate building blocks of theformula VIIIb in the DNA reaction cycle to incorporate themethylphosphonate bridges _(Me).

Example 21 5′-C_(S),_(S)A_(S),_(S)C GT_(S),_(S)T GAG (but)Ggg cat-hex(c-myc antisense)

(Example of Structural Type XIIa in Scheme 1; _(S,S) Means aPhosphorodithioate Bridge).

The synthesis takes place in analogy to the description in Example 17but the building block VIIId is used to incorporate the dithioatebridges, and the oxidation at these sites (step 20) is carried out withTETD.

Example 22 N-cga g(5′NH-A)A CAT CA (Oeg(t))ggt cg-hex (c-fos antisense)

(5′NH-A Means 5′-amino-5′-deoxyadenylate; see Example 9 for Explanationof Oeg(t))

The synthesis takes place in analogy to the description in Example 18with, after completion of the DNA synthesis, in analogy to Example 13condensation on of a 5′-aminonucleotide which permits conjugation of thesecond PNA part. Thus, firstly six PNA synthesis cycles are carried outand then the linker building block from Example 9 is coupled on. Thenseven DNA synthesis cycles are carried out, using the building block ofthe formula VIIIf in the last cycle. After a further four PNA synthesiscycles have been carried out, the elimination from the support andfurther working up are carried out as described in Example 19.

Example 23 F-cga g(5′NH-A)A CAT CAT GGT_(S)C_(S)G-O—CH₂CH(OH)CH₂—O—C₁₆H₃₃

(5′NH-A Means 5′-amino-5′-deoxyadenylate; F a fluorescein Residue on theamino terminus of the PNA and _(S) a Phosphorothioate Bridge)

The synthesis takes place in analogy to the description in Example 19but starting from a CPG support onto which the glycerol hexadecyl etheris bound. After 12 DNA synthesis cycles have been carried out, thelinker building block VIIIf is condensed on. After four PNA synthesiscycles have been carried out and the terminal Mmt group has beeneliminated, it is possible to react the free amino group quantitativelywith a 30-fold excess of fluorescein isothiocyanate (FITC).

Example 24 3′-CCC TCT T-5′-(PEG)(PEG)-(Oeg(t))tg tgg g-hex

(PEG Means a Tetraethylene Glycol Phosphate Residue)

The synthesis in respect of the PNA part takes place in analogy to thedescription in Example 17. After six PNA units have been condensed on,the (Mmt-Oeg(t)-OH) from Example 9 is coupled on. Then as linkerinitially the tetraethylene glycol derivative of the formula XV iscondensed on twice as described in the DNA synthesis cycle before thesynthesis of the DNA part with reversed orientation (from 5′ to 3′) iscarried out. For this purpose, in place of the nucleoside3′-phosphoramidites in each case the corresponding nucleoside5′-phosphoramidites of the formula XIV, which are commerciallyavailable, are used in step 16 in the DNA synthesis cycles. Furtherdeprotection and working up take place as described in Example 17.

Example 25 N-ccc tct t-(C6-link)(PEG)-3′-AAG AGG G-5′

(PEG Means a Tetraethylene Glycol Phosphate Residue; C6-Link is a6-Aminohexanol Phosphate Residue)

The synthesis takes place in analogy to the description in Example 17(DNA synthesis cycle) but starting from a CPG support to which3′-O-Dmt-deoxyguanosine is bound via a 5′-O-succinate group. After sixDNA units have been condensed on using the building blocks of theformula XIV, initially the tetraethylene glycol derivative of theformula XV is condensed on once as linker before coupling thephosphoramidite of the formula XVI to introduce C6-link. The PNA part isthen synthesized on as in Example 17 (PNA synthesis cycle). Furtherdeprotection and working up take place as described in Example 19.

Example 26 5′-TTT TTT TTT (but) ttt ttt-hex

The synthesis takes place in analogy to the description in Example 17.Before the product is cleaved off the support and deprotected, half thesupport-bound DNA/PNA hybrid is taken for fluorescence labeling (Example27). The other half is deprotected and worked up as described in Example17.

Example 27

(FAM is Fluorescein Residue)

5′-FAM-TTT TTT TTT (but) ttt ttt-hex

The support-bound DNA/PNA hybrid from Example 26 is fluorescence labeledby carrying out steps 13 to 20 as described in Example 17 using thefluorescein phosphoramidite from Applied Biosystems in step 16.

Example 28 5′-GGG GGG GGG (but) ttt ttt-hex

The synthesis takes place in analogy to the description in Example 17.Before the product is cleaved off the support and deprotected, half thesupport-bound DNA/PNA hybrid is taken for fluorescence labeling (Example29). The other half is deprotected and worked up as described in Example17. The title compound binds as triplex-forming oligonucleotide withhigh affinity to a DNA double strand which contains the homopurine motif5′-AAA AAA GGG GGG GGG-3′.

Example 29

(FAM is Fluorescein Residue)

5′-FAM-GGG GGG GGG (but) ttt ttt-hex

The support-bound DNA/PNA hybrid from Example 28 is fluorescence labeledby carrying out steps 13 to 20 as described in Example 17 using thefluoresceine phosphoramidite from Applied Biosystems in step 16.

Example 30 Biotin-C_(Phe)G_(Phe)A GAA cat cat(5′NH-G)G(Ome)U(Ome)C(Ome)-G(ome)-VitE (c-fos antisense)

(N(Ome) means a nucleotide unit N with a 2′-O-methoxy group; _(Phe)means a phenylphosphonate bridge; 5′NH-G means5′-amino-5′-deoxyguanylate).

The synthesis takes place in analogy to the description in Example 17starting from CPG which is loaded with vitamin E (MacKellar et al.(1992) Nucleic Acids Res, 20(13), 3411-17) and coupling the buildingblock of the formula VIIIe four times after the DNA synthesis cycle.After the 5′-aminonucleotide building block of the formula VIIIf hasbeen coupled on, six PNA units are condensed on after the PNA synthesiscycle. After neutralization, the phosphoramidite is coupled to the aminogroup by a known method, and the DNA synthesis cycle is repeatedappropriately to assemble the DNA part, using in the case of thephenylphosphonate bridges the building blocks of the formula VIIIc instep 16. Lastly the end group is coupled on using the biotinphosphoramidite from Glen Research. After the synthesis is complete, theoligomer is deprotected as described in Example 19, eliminating thedimethoxytrityl group at the end by treatment with 80% strength aceticacid at 22° C. for 2 hours.

Example 31 A CAT CA (Oeg(t)) ggt cg-hex (c-fos antisense)

(See Example 9 for Explanation of Oeg(t))

The synthesis takes place in analogy to the description in Example 18.In this case, firstly five PNA synthesis cycles are carried out and thenthe linker building block Oeg(t) from Example 9 is coupled on. Then sixDNA synthesis cycles are carried out. Subsequently, the elimination fromthe support and the further working up are carried out as described inExample 18.

Example 32 A TAA TG (Oeg(t)) tct cg-hex (Control Oligomer for c-fos)

The synthesis takes place in analogy to the description in Example 18.In this case, firstly five PNA synthesis cycles are carried out and thenthe linker building block Oeg(t) from Example 9 is coupled on. Then sixDNA synthesis cycles are carried out. Subsequently, the elimination fromthe support and the further working up are carried out as described inExample 18.

Example 33 a cat cat ggt cg-hex (c-fos Antisense)

This pure PNA oligomer was prepared as reference compound in analogy toExample 18 but with the exception that twelve PNA cycles were carriedout. Deprotection of the exocyclic amino protective groups is carriedout in ammoniacal solution (5 hours at 55° C.). Only then is themonomethoxytrityl group eliminated by treatment with 80% strength aceticacid at 22° C. for 2 hours.

Example 34 A (5-hexy-C)A(5-hexy-U) (5-hexy-C)A (Oeg(t)) ggt cg-hex(c-fos antisense)

(See Example 9 for Explanation of Oeg(t); 5-hexy-C Means5-hexynylcytidine, 5-hexy-U Means 5-hexynyluridine)

The synthesis takes place in analogy to the description in Example 31but using in place of the normal pyrimidine phosphoramidites thecorresponding 5-hexynylpyrimidine nucleoside phosphoramidites in thecondensation reaction.

Example 35

(PAM is Fluorescein Residue)

5′-FAM-TT (but) ttt ttt-hex

The synthesis of this PNA/DNA oligomer takes place in analogy to thedescription in Example 27 although only two thymidylate units arecondensed on.

Example 36 taa tac gac tca cta (5′HN-T)

(5′HN-T means 5′-amino-5′-deoxythymidine)

This PNA/DNA oligomer which is composed of 15 PNA units and onenucleoside unit was synthesized as primer for the DNA polymerasereaction. This entails starting from a solid phase support(aminoalkyl-CPG) to which the5′-monomethoxytritylamino-5′-deoxythymidine is bound via its 3′-hydroxylgroup as succinate. After elimination of the monomethoxytrityl groupwith 3% TCA in dichloromethane, 15 PNA cycles are carried out asdescribed in Example 17. Deprotection of the exocyclic amino protectivegroups is carried out in ammoniacal solution (5 hours at 55° C.). Onlythen is the monomethoxytrityl group eliminated by treatment with 80%strength acetic acid at 22° C. for 2 hours. A PNA/DNA oligomer with afree 3′-hydroxyl group, which is used as primer for a DNA polymerase(Klenow) is obtained.

Example 37 p_(S)-rA(2′5′)rA(2′5′)rA(2′5′)rA-spacer-(Oeg(t)tc ctc ctgcgg-hex

(p_(S) Means a 5′-Thiophosphate; Spacer Means a Triethylene GlycolPhosphate; rA is a Riboadenylate; (2′5′) Means that the InternucleotideLinkage is from 2′ to 5′ in the Ribose)

The synthesis of this compound takes place in analogy to the descriptionin Example 18 by initially condensing on 14 PNA units. After the linkerbuilding block Mmt-Oeg(t)-OH from Example 9 has been introduced underthe conditions described in step 5, the Mmt group is eliminated with 3%TCA, and the spacer is introduced with the aid of the commerciallyavailable Dmt-O—(CH₂CH₂O)₃—O—P(—OCH₂CH₂CN)N(i-C₃H₇)₃ spacerphosphoramidite (from Eurogentech; Brussels). The (2′5′)-linkedtetradenylate is synthesized on as described in Example 17 using thecommercially availableN⁶-benzoyl-5′-O-Dmt-3′-O-tertbutyldimethylsilyladenosine 2′-O-cyanoethyldiisopropylaminophosphoramidite (from Milligen, Bedford, USA), extendingthe condensation time to 2×5 min. The stronger activator5-ethylthiotetrazole is used in place of tetrazole in the couplingreaction. After elimination of the last Dmt group, the oligomer isphosphitylated on the 5′-OH group withbis(β-cyanoethyloxy)diisopropylaminophosphine. Oxidation with TETD anddeprotection with ammonia and desilylation with fluoride result in thetitle compound, which stimulates RNase L.

Example 38 p_(S)-Co(2′5′)Co(2′5′)Co(2′5′)Co-spacer-(Oeg(t))tc ctc ctgcgg-hex

(p_(S) Means a 5′-Thiophosphate; Spacer Means a Triethylene GlycolPhosphate; Co is Cordycepin (3′deoxyadenosine); (2′5′) Means that theInternucleotide Linkage is from 2′ to 5′)

The synthesis is carried out in analogy to Example 37 but in place ofthe N⁶-benzoyl-5′-O-Dmt-3′-O-tert-butyldimethylsilyladenosine2′-O-cyanoethyl diisopropylaminophosphoramidite, the correspondingN⁶-benzoyl-5′-O-Dmtcordycepin 2′-O-cyanoethyldiisopropylaminophosphoramidite (from Chemogen, Konstanz) is used, andthe fluoride treatment is omitted.

Example 39 5′-GG GGG GGG (Oeg(t)) ttt ttt ttt-hex

The synthesis takes place in analogy to the description in Example 18,following nine PNA couplings by condensation on of the linker buildingblock Mmt-Oeg(t)-OH from Example 9 under the conditions described instep 5, which permits subsequent condensation of eight guanylateresidues. The resulting PNA/DNA oligomer binds with high affinity in theantiparallel orientation as triplex-forming oligonucleotide todouble-stranded DNA which has the sequence 5′. . . AAAAAAAAAAGGGGGGGG .. . 3′.

Example 40 Characterization of the PNA/DNA Hybrids

The characterization takes place with the aid of HPLC, polyacrylamidegel electrophoresis (PAGE) and negative ion electrospray massspectrometry (ES−MS⁻). The products are purified as described above andthereafter show in the PAGE (20% acrylamide, 2% bisacrylamide and 7 Murea) a single band. The RPLC takes place on RP-18 reversed phasecolumns from Merck (eluent A: water with 0.1% TFA, B:water/acetonitrile=1:4; linear gradient) or on a PA-100 column fromDionex (eluent A: 20 mM NaOH and 20 mM NaCl; B: 20 mM NaOH and 1.5 MNaCl; linear gradient). For the ES−MS⁻, the PNA/DNA hybrids areconverted by ammonium acetate precipitation or other metathesis into theammonium salts. Sample introduction takes place from a solution inacetonitrile/water (1:1) containing 5 OD₂₆₀/ml oligomer. The accuracy ofthe method is about ±1.5 Dalton.

Example 41 Determination of Cellular Uptake and Stability afterRadioactive Labeling

Radioactive Labeling:

A generally applicable labeling with ³⁵S comprises carrying out at leastone oxidation in the DNA synthesis cycle (step 20 in Example 17) for thesynthesis of the DNA part using elemental sulfur-35. PNA/DNA hybridswhich have a free 5′-hydroxyl group can be labeled with ³²P or ³⁵S withthe aid of polynucleotide kinase by methods known per se. PNA/DNAhybrids which carry a free 3′-hydroxyl group can be labeled in a knownmanner with 3′-terminal transferase. As an example, the 5′-labeling ofthe DNA part is described here: the PNA/DNA hybrid with a free5′-hydroxyl group (500 pmol) from Example 17, 18 or 26 is dissolved in425 μl of water, and this solution is heated to 90° C. and rapidlycooled. Then 50 μl of 10×kinase buffer and 50 μl of ³²P-gamma-ATP (6,000Ci/mmol) or ³⁵S-gamma-ATP are added, and the mixture is incubated at 37°C. for 1 hour. The reaction is stopped by adding 0.5 M EDTA solution.Desalting takes place with the aid of an NAP^(R) column from Pharmacia.

Determination of Cellular Uptake:

Vero cells are incubated in DMEM, 5% FCS, in 96-well microtiter platesat 37° C. for 24 hours. After removal of the medium, the cells arewashed twice with serum-free DMEM. The radioactively labeled oligomer(10⁶ cpm) is diluted with unlabeled oligomer to a concentration of 10 μMin serum, and the cells are incubated at 37° C. therewith. 150 μlportions are removed after 1, 7 and 24 hours (called “supernatant 1”).The cells in the wells of the microtiter plates are washed 7 times with300 μl of fresh medium, and the combined washing media (called“supernatant 2”) are measured in a scintillation counter. Then 100 μl oftrypsin solution are added, 30 seconds are allowed to elapse, and thesupernatant is aspirated off. The cells are detached from the plate byincubating at 37° C. for 3 min. The detached cells are transferred into1.5 ml Eppendorf tubes and centrifuged at 2,000 rpm for 6 minutes(“supernatant 3”). Supernatants 1 (5 μl), 2 and 5 (0.5 ml) are eachmeasured separately in a scintillation counter. From this is calculatedthe uptake of oligomer in pmol per 100,000 cells, with supernatant 3representing the cell-bound oligomer fraction and the total ofsupernatants 1 and 2 representing the non-cell-bound oligomer fraction.

Results: Cellular uptake in pmol Incubation time of oligomer/10⁵ cellsin hours PNA/DNA hybrid DNA 1 0.25 0.36 7 0.54 0.57 24 0.75 0.78Investigation of the Stability of the Oligomer in Medium ContainingCells:

Supernatant 1 (10 μl) is mixed with 5 μl of 80% formamide (withXylenecyanol and bromphenolblue), heated to 95° C. (5 minutes) andloaded onto a polyacrylamide gel (20% acrylamide, 7 M urea). Afterdevelopment of the gel in the electric field, the bands on the gel areassigned by autoradiography to the “stable oligomer”, and the missingbands to the “degraded oligomer”.

The PNA/DNA oligomer from Example 26 is 69% stable after an incubationtime of 24 hours; the DNA oligomer is 3% stable.

The PNA/DNA oligomer from Example 31 has a half-life of 32 h under theseconditions, whereas the corresponding DNA oligonucleotide has ahalf-life of about 2 h.

Example 42 Determination of Cellular Uptake by Fluorescence Labeling

COS cells are allowed to grow to confluence in Dulbecco's MEMsupplemented with 10% FCS in 5 cm Petri dishes. The cells are washedtwice with serum-free DMEM. A sterile needle is used to scratch an areaof about 1 cm² in the middle of the Petri dish. The PNA/DNA oligomersolution (0.1 mM) to be investigated is applied to this area. Incubationis carried out at 37° C. under a CO₂ atmosphere. The cells areinvestigated by fluorescence microscopy after 2, 4 and 16 hours. Forthis, the cells are washed four times with serum-free DMEM, covered witha glass slide and assessed under a fluorescence microscope or by phasecontrast. A fluorescence-labeled PNA (without DNA part) F-(but)-ttttttt-hex was investigated as comparison for the PNA/DNA hybrid molecules.After the cells had been incubated with this PNA for two hours, >90% ofthe cells show signs of pronounced morphological changes and cell death.Most of the cells exhibit pronounced vacuolization. The plasma membrane,the cytosol and the nucleus show no uptake of PNA. After incubation withthe pure PNA for a further two hours, all the cells have died. Thesituation is different with the DNA/PNA oligomers according to theinvention. After incubation of the cells with the DNA/PNA oligomers foronly two hours the cells show punctiform intracellular distribution ofthe PNA/DNA oligomers. The cells suffer no cell death even afterprolonged incubation.

Example 43 Determination of the Melting Temperatures

The melting temperatures are determined using an HP 8452A diode arrayspectrophotometer, an HP 89090A Peltier element and the HP temperaturecontrol software Rev.B5.1 (from Hewlett Packard). Measurements arecarried out in 0.5° C./min steps in 10 mM HEPES and 140 mM NaCl (pH 7.5)as buffer. The oligomer concentration is 0.5 to 1 OD₂₆₀ per ml.

Result for the product from Example 17 or 18 (T_(M) with DNA)

5′-ATC GTC GTA T(Oeg(t))a gtc-hex T_(M) = 51.5° C. 3′-TAG CAG CAT A   AT CAG-5′ antiparallel 5′-ATC GTC GTA T(Oeg(t))a gtc-hex T_(M) < 20° C.5′-TAG CAG CAT A   A T CAG-3′ parallel 5′-ATC GTC GTA TT(but)a gtc-hexT_(M) = 51.0° C. 3′-TAG CAG CAT AA   T CAG-5′ antiparallel 5′-ATC GTCGTA TTA GTC-3′ T_(M) = 50.5° C. 3′-TAG CAG CAT AAT CAG-5′ DNA · DNAantiparallel 5′-ATC GTC GTA TT(but)a gtc-hex T_(M) < 20° C. 5′-TAG CAGCAT AA   T CAG-3′ parallel T_(M) with RNA Sequence T_(M) with DNA (T= U) 5′-ACA TCA TGG TCG-3′ DNA ap 50.7° C. 48.6° C. 3′-TGT AGT ACCAGC-5′ 5-ACA TCA tgg tcg-3′ (PNA/DNA) ap 54.5° C. 54.7° C. 3′-TGT AGTACC AGC-5′ 5′-ACA TCA tgg tcg-3′ (PNA/DNA) p 20° C.   <20° C.    3′-TGTAGT ACC AGC-3′ 5′-aca tca tgg tcg-3′ PNA ap 58.8° C. 66.6° C. 3′-TGT AGTACC AGC-5′ 5′-aca tca tgg tcg-3′ PNA p 46.3° C. 44.8° C. 5′-TGT AGT ACCAGC-3′ 5′-ACA TCA TGG TCG-3′ S-DNA ap 46.7° C. 43.8° C. 3′-TGT AGT ACCAGC-5′

TGG TCG means a DNA part in which all internucleotide linkages are inphosphorothioate form. See page 5 for definition of p and ap.

Example 44 Tests for Antiviral Activity

The antiviral activity of the test substances on varioushuman-pathogenic Herpesviruses is investigated in a cell culture testsystem. For the experiment, monkey kidney cells (Vero, 2×10⁵/ml) areinoculated in serum-containing Dulbecco's MEM (5% fetal calf serum FCS)in 96-well microtiter plates and incubated at 37° C. and 5% CO₂ for 24h. The serum-containing medium is then aspirated off and the cells arerinsed twice with serum-free Dulbecco's MEM (−FCS). The test substancesare prediluted in H₂O to a concentration of 600 μM and stored at −18° C.Further dilution steps in Dulbecco's minimal essential medium (MEM) arecarried out for the test. 100 μl portions of the individual testsubstance dilutions are added together with 100 μl of serum-freeDulbecco's MEM (−FCS) to the rinsed cells. After incubation at 37° C.and 5% CO₂ for 3 h, the cells are infected with Herpes simplex virustype 1 (ATCC VR733, HSV-1 F-strain) or with Herpes simplex virus type 2(ATCC VR734, HSV-2 G-strain) in concentrations at which the cell lawn iscompletely destroyed within 3 days. The infection concentration forHSV-1 is 500 plaque-forming units (PFU) per well, and for HSV-2 it is350 PFU/well. The test mixtures then contain test substance inconcentrations of 80 μM to 0.04 μM in MEM supplemented with 100 U/mlpenicillin G and 100 mg/l streptomycin. All the experiments are carriedout as duplicate determination with the exception of the controls whichare carried out eight times per plate. The test mixtures are incubatedat 37° C. and 5% CO₂ for 17 h. The cytotoxicity of the test substancesis determined after a total incubation time of 20 h by microscopicinspection of the cell cultures. The maximum tolerated dose (MTD) isdefined as the highest concentration of product which, under the statedtest conditions, does not yet cause microscopically detectable celldamage. Subsequently FCS is added to a final concentration of 4%, andincubation is continued at 37° C. and 5% CO₂ for 55 h. The untreatedinfection controls then show a complete cytopathic effect (CPE). Aftermicroscopic inspection of the cell cultures they are then stained withneutral red in accordance with the vital staining method of Finter(1966). The antiviral activity of a test substance is defined as theminimum inhibitory concentration (MIC) which is needed to protect 30-60%of the cells from the cytopathogenic effect caused by the virus. Theactivity of the PNA/DNA chimeras is in each case better than that of thecorresponding DNA oligomers or PNA oligomers.

Example 45 Determination of the in vivo Activity: Inhibition of c-FosProtein Expression in the Rat

The determination takes place as described (Sandkühler et al. (1991) in:Proceedings of the VIth World Congress on Pain, Charlton and Woolf,Editors; Elsevier, Amsterdam; pages 313-318) by superfusion of thespinal cord. After laminectomy of a barbiturate-anesthetizedSprague-Dawley rat, a two-chamber container is formed from silicone toreceive the antisense oligomer. One chamber is filled with the antisensePNA/DNA derivative, while the other chamber is filled with the controloligomer (concentration of each 75 μM). The superfusate is exchanged ineach case after one hour. After superfusion for 6 hours, c-fosexpression is stimulated by heat treatment (52° C.) of the rear legs.Inhibition of c-fos expression can be demon-stratedimmunohistochemically on appropriate tissue section samples. The c-fosantisense oligonucleotide from Example 31 brings about greaterinhibition of c-fos expression than does the corresponding DNAoligonucleotide and the corresponding PNA oligomer from Example 33.

Example 46 RNase H Assay

To determine the RNase H activity, 1.3 OD of the PNA/DNA oligomer to beinvestigated are heated with 0.5 OD of the complementary RNA sequence(target sequence) dissolved in 50 μl of autoclaved water, treated withDEPC (diethyl pyrocarbonate), at 80° C. for 5 minutes and subsequentlycooled to 37° C. within 15 minutes. This results in initial denaturationof both oligomers which, after cooling, form a nucleic acid doublestrand in sequence-specific manner.

For the assay, this RNA.PNA/DNA duplex is incubated with 10 μl of RNaseH 10× buffer, 1 μl of dithiothreitol (DTT) and 2 μl (corresponding to 10u) of RNase H supplied by USB. The incubation mixture is made up withautoclaved, DEPC-treated water to the required total volume of 100 μl.The samples are incubated as 37° C. For the kinetic investigation, 20 μlportions of the solution were removed after 0, 2 min, 10 min and 1 h,heated at 95° C. for 5 minutes and frozen at −70° C. until analyzed. Theinvestigation of the RNase H cleavage of RNA takes place by gelelectrophoresis. It emerged that PNA/DNA hybrids which containdeoxyribonucleotide building blocks activate RNase H, with cleavage ofthe complementary RNA strand whereas the PNA/DNA oligomer emergesundamaged from the reaction. The cleavage reaction with the PNA/DNAoligomer takes place somewhat more slowly than with a correspondingoligodeoxyribonucleotide of equal length and sequence.

Example 47 Preparation of an HeLa Cell Extract with RNase L Activity

An HeLa cell extract was prepared in order to stimulate the activity ofcellular endoribonuclease L by the 2′,5′-tetraadenylate-PNA/DNAconjugates. For this purpose, 35 bottles were each charged with 20 ml ofmedium containing Dulbecco's MEM (mimimal essential medium) and 10% FCS(fetal calf serum). The cells can be harvested after trypsin treatment.4 ml of cell harvest are obtained after centrifugation at 1,000 rpm.This is initially made up with 4 ml of water and, after 3 minutes, 4 mlof buffer A (5.48 g of HEPES; 15.5 g of KCl; 2.488 g of Mg acetate;1,232 μl of 2-mercaptoethanol ad 1 l with water) are added in order tolyze the cells. The solution is then centrifuged at 30,000 rpm (about100,000 g) in an ultracentrifuge at 0° C. for 30 minutes. Thesupernatant from 8 ml of cell extract is removed and stored at −20° C.for the following investigations.

Example 48 Investigation of Activiation of RNase L

For investigation of this extract for endonuclease L, initially 0.3 ODof the RNA target sequence is heated with the particular PNA/DNAoligomers at 80° C. for 5 minutes and subsequently cooled to 37° C. forthe hybridization. The duplex is mixed with 20 μl of the extract, 1.2 μlof glycerol and RNase L buffer and incubated at 37° C. The total volumeis then 70 μl. For the kinetic investigations, samples are removed bypipette at the times of 0, 20 and 60 minutes and heated at 95° C. for 5minutes to denature the enzymes. The samples are lyophilized in aspeedvac and analyzed by gel electrophoresis. ThePNA-2′,5′-tetraadenylate conjugates and tetracordycepin analogs activatecellular RNase L, whereas corresponding compounds without thetetraadenylate part do not stimulate RNase L.

Example 49 DNA Polymerase Reaction

The following 81-mer oligodeoxynucleotide is used as template for theDNA polymerase reaction:

5′-GCC CCA GGG AGA AGG CAA CTG GAC CGA AGG CGC TTG TGG AGA AGG AGT TCATAG CTG GGC TCC CTA TAG TGA GTC GTA TTA-3′

The sequence of the PNA/DNA primer is:

-   H-taa tac gac tca cta (5NH-T)-OH 3′.

A corresponding oligodeoxynucleotide of the sequence 5′-TAA TAC GAC TCACTA TAG-3′ is used as control primer.

The primer (0.15 nmol) and the template (0.15 nmol) in 5 μl of 10×PCRbuffer (500 m KCl, 100 mM tris-HCl, pH 9, 1% Triton X-100, 15 mM MgCl₂)are diluted with 35 μl of water and hybridized by heating to 95° C. andcooling. Then 10 μl of 2 mM dNTP mixture (nucleoside 5′-triphosphates)and 3 μl of DNA polymerase (Klenow fragment) are added, and the mixtureis incubated at 22° C. and 37° C. for 0.5 hour each. The reactionsolution is then analyzed on a 10% polyacrylamide gel (with 1% bis).pBR322/HaeIII digestion is loaded as marker. The reaction with thecontrol primer shows a double-stranded DNA fragment with the expectedsize relative to the marker, whereas the product from the PNA/DNA primermigrates somewhat more quickly. In both cases the double strand migratesconsiderably faster than the template single strand in the gelelectrophoresis.

1. A gene probe assay for the determination of an oligo- orpolynucleotide target (RNA or DNA), wherein the gene probe assay ishomogeneous or heterogeneous and comprises hybridizing a gene probe withthe oligo- or polynucleotide target, wherein the gene probe is apolyamide-oligonucleotide derivative of the formula Ia:

wherein q=r=s=1 t =zero R² is hydrogen, hydroxyl, C₁-C₁₈-alkoxy,halogen, azido or amino; B is, independently of one another, a basecustomary in nucleotide chemistry, or a prodrug form thereof, Nu is aradical of the formulae IIa or IIb

in which R² and B are as defined above; U is hydroxyl, mercapto,C₁-C₁₈-alkyl, C₁-C₁₈-alkoxy, C₆- C₂₀-aryl, C₆-C₁₄-aryl-C₁-C₈-alkyl, NHR³or NR³R⁴, V is oxy, thio or imino; W is oxo or thioxo; Y is oxy, thio,methylene or imino; D is a radical of the formula III

in which B is as defined above; n is zero to 20; p is zero to 20; Li₁and Li₂ are each, independently of one another, a structure of theformula V[(V′)-(G)-(G′)]_(ε)  (V) where, independently of one another, ε is 1 to5, V′ is oxygen, NH, a bond or a radical of the formula VI

in which U, V, W and Y are as defined above; G can be C₁-C₁₂-alkanediyl,where alkanediyl can optionally be substituted by halogen, amino,hydroxyl, C₁-C₁₈-alkyl, C₁-C₁₈-alkoxy, C₆-C₁₄-aryl, orC₆-C₁₄-aryl-C₁-C₁₈-alkyl; C₆-C₁₄-aryl-di-C₁-C₁₂-alkanediyl, or a groupof the formula (CH₂CH₂O)_(δ)CH₂CH₂ in which δ can be 1 to 11; or a bond;and G′ is oxy, thio, imino, —C(O)—, —C(O)NH—, a bond or a radical of theformula VI in which U, V, W and Y are as defined above; and F and F′ arelinked by a bond and/or F is R⁰-(A)_(k)-V- and F′ in formula Ia is-(Q)¹- R¹ and in formula Ib is V¹- (A)₁-R¹, where R⁰ is hydrogen,C₁-C₁₈-alkanoyl, C₁-C₁₈-alkoxycarbonyl, C₃-C₈-cycloalkanoyl,C₇-C₁₅-aroyl, C₃-C₁₃-heteroaroyl or a group which, in the hybridizationof the oligonmer onto the target nucleic acid, crosslinks with, orcleaves the target nucleic acid, or if k is zero, R⁰ is hydrogen ortogether with V is a radical of the formula VII

in which Z and Z′ are, independently of one another, hydroxyl, mercapto,C₁-C₂₂-alkoxy, C₁-C₁₈-alkyl, C₆-C₂₀-aryl, C₆-C₁₄-aryl-C₁-C₁₈-alkyl,C₁-C₂₂-alkylthio, NHR³, NR³R⁴, or a group wherein intracellular uptakeof the oligomer occurs or which labels a DNA probe or, in thehybridization of the oligomer onto the target nucleic acid, crosslinkswith, or cleaves the target nucleic acid, and wherein R³, R⁴, V and Ware as defined above; R¹ is hydrogen or Q^(o) where R¹ is always onlyhydrogen when at the same time 1 is zero and in formula Ia t is zero ands is 1 and Li₁ is a structure of the formula V with V′=bond, G =bond,ε=1 and G′=oxy, thio, imino or a radical of the formula VI with U =ZQ^(o) is hydroxyl, OR′, NH₂, NHR″ with R′=C₁-C₁₈-alkyl andR″=C₁-C₁₈-alkyl, C₁-C₁₈-aminoalkyl, C₁-C₁₈-hydroxyalkyl; V is as definedabove; V¹ is a bond or V, where in F′ only in formula Ib with q=zero andr=1 V¹ is always a bond; k is zero to 10; l is zero to 10; with theproviso that a) if in the compound of the formula Ia t is zero and s is1, and Li₁ is (V′)−(G)−(G′) with V′=a compound of the formula VI, G=C₂-C₁₂-alkylene and G′=CO, in F′=-(Q)₁-R¹ 1 is zero to 10 and R¹ isQ^(o); and b) if in the compound of the formula Ia s=t=zero, Li₂ is abond; where each nucleotide can be in its D or L configuration, and thebase can be in the α or β position.
 2. The gene probe assay of claim 1wherein said B in said polyamide-oligonucleotide derivative is chosenfrom the group consisting of adenine, cytosine, thymine, guanine,uracil, inosine purine, 2,6 di-aminopurine, 7-deazaadenine,7-deazaguanine, N⁴,N⁴-ethanocytosine, N⁶,N⁶-ethano-2,6-diaminopurine,pseudoisocytosine, 5-methylcytosine, 5-fluorouracil,5-(C3-C6)-alkynyluracil, 5-(C3-C6)-alkynylcytosine and prodrug formsthereof.
 3. The gene probe assay of claim 1 wherein said A and Q,independently of one another, are chosen from the group consisting ofglycine, leucine, histidine, phenylalanine, cysteine, lysine, arginine,aspartic acid, glutamic acid, proline,tetrahydroisoquinoline-3-carboxylic acid, octahydroindole-2 carboxylicacid, N-(2- aminoethyl)-glycine.
 4. The gene probe assay of claim 1,wherein the gene probe further comprises a detectable label.